Materials comprising and methods of preparation and use for ribosome-inactivating proteins

ABSTRACT

The present invention provides purified and isolated polynucleotides encoding Type I ribosome-inactivating proteins (RIPs) such as gelonin and analogs of the RIPs having a cysteine available for disulfide bonding to targeting molecules. Vectors comprising the polynucleotides and host cells transformed with the vectors are also provided. The RIPs and RIP analogs are particularly suited for use as components of cytotoxic therapeutic agents of the invention which include gene fusion products and immunoconjugates. Cytotoxic therapeutic agents or immunotoxins according to the present invention may be used to selectively eliminate any cell type to which the RIP component is targeted by the specific binding capacity of the second component of the agent, and are suited for treatment of diseases where the elimination of a particular cell type is a goal, such as autoimmune disease, cancer and graft-versus-host disease.

This application is a continuation-in-part of co-pending U.S. patentapplication Ser. No. 07/901,707, filed Jun. 19, 1992, which in turn is acontinuation-in-part of U.S. patent application Ser. No. 07/787,567,filed Nov. 4, 1991, now abandoned.

BACKGROUND

The present invention relates, in general, to materials useful ascomponents of cytotoxic therapeutic agents. More particularly, theinvention relates to polynucleotides encoding ribosome-inactivatingproteins, to polynucleotides encoding analogs of ribosome-inactivatingproteins specifically modified for conjugation to targeting moleculesand to gene fusions of polynucleotides encoding ribosome-inactivatingproteins to polynucleotides encoding targeting molecules.

Ribosome-inactivating proteins (RIPs) comprise a class of proteins whichis ubiquitous in higher plants. RIPs have also been isolated frombacteria. RIPs are potent inhibitors of eukaryotic protein synthesis.The N-glycosidic bond of a specific adenine base is hydrolyticallycleaved by RIPs in a highly conserved loop region of the 28S rRNA ofeukaryotic ribosomes thereby inactivating translation.

Stirpe et al., FEBS Lett., 195(1,2), 1-8 (1986) groups plant RIPs intotwo types. Type I proteins each consist of a single peptide chain havingribosome-inactivating activity, while Type II proteins each consist ofan A-chain, essentially equivalent to a Type I protein, disulfide-linkedto a B-chain having cell-binding properties. Gelonin, dodecandrin,tricosanthin, tricokirin, bryodin, Mirabilis antiviral protein (MAP),barley ribosome-inactivating protein (BRIP), pokeweed antiviral proteins(PAPs), saporins, luffins and momordins are examples of Type I RIPs,while ricin and abrin are examples of Type II RIPs. Amino acid sequenceinformation is reported for various ribosome-inactivating proteins. Itappears that at least the tertiary structure of active sites isconserved among Type I RIPs, bacterial RIPs and A-chains of Type II RIPsand, in many cases, primary structure homology is also found. Ready etal., J. Biol. Chem., 259(24), 15252-15256 (1984) and other reportssuggest that the two types of RIPs are evolutionarily related.

Separated from their natural environment, Type I plantribosome-inactivating proteins may be particularly suited for use ascomponents of cytotoxic therapeutic agents. A RIP may be conjugated to atargeting agent that will deliver the RIP to a particular cell type invivo in order to selectively kill those cells. Typically, the targetingagent (e.g., an antibody) is linked to the toxin by a disulfide bondwhich is reduced in vivo allowing the protein toxin to separate from thedelivery antibody and become active intracellularly. Another strategyfor producing a cytotoxic agent is to express a gene encoding a RIPfused to a gene encoding a targeting moiety. The resulting proteinproduct is a single polypeptide containing an RIP linked to, forexample, at least one chain of an antibody. A variety of gene fusionproducts including protein toxin sequences are discussed in a recentreview by Pastan et al., Science, 254, 1173-1177 (1991).

Because some RIPs, such as the Type I RIP gelonin, are only availablefrom scarce plant materials, it is desirable to clone the genes encodingthe RIPs to enable recombinant production of the proteins. It is alsodesirable to develop analogs of the natural proteins which may be easilyconjugated to targeting molecules while retaining their naturalbiological activity because most Type I RIPs have no natural sites (i.e.available cysteine residues) for conjugation to targeting agents.Alternatively, it is desirable to develop gene fusion products includingType I RIPs as a toxic moiety and antibody substances as a targetingmoiety.

There thus exists a need in the art for cloned genes encoding Type IRIPs, for analogs of Type I RIPs which may be easily conjugated totargeting molecules and for gene fusion products comprising Type I RIPs.

SUMMARY OF THE INVENTION

The present invention provides purified and isolated polynucleotidesencoding Type I RIPs, Type I RIPs having a cysteine available fordisulfide bonding to targeting molecules and fusion products includingType I RIPs. Vectors comprising the polynucleotides and host cellstransformed with the vectors are also provided.

A purified and isolated polynucleotide encoding natural sequencegelonin, and a host cell including a vector encoding gelonin of the typedeposited as ATCC Accession No. 68721 are provided. Further provided area purified and isolated polynucleotide encoding natural sequence barleyribosome-inactivating protein and a purified and isolated polynucleotideencoding momordin II.

Analogs of a Type I plant RIP are defined herein as non-naturallyoccurring polypeptides that share the ribosome-inactivating activity ofthe natural protein but that differ in amino acid sequence from thenatural protein. Preferred analogs according to the present inventionare analogs of Type I plant RIPs each having a cysteine available fordisulfide bonding located at a position in its amino acid sequence fromthe position corresponding to position 251 in SEQ ID NO: 1 to thecarboxyl terminal position of the analog. Other preferred analogsaccording to the invention are Type I RIPs each having a cysteineavailable for disulfide bonding at a position in the analog that is onthe surface of the protein in its natural conformation and that does notimpair native folding or biological activity of theribosome-inactivating protein. Analogs of bacterial RIPs are alsocontemplated by the present invention.

The present invention provides an analog of a Type Iribosome-inactivating protein, which analog has a cysteine available forintermolecular disulfide bonding at an amino acid position correspondingto a position not naturally available for intermolecular disulfidebonding in the Type I ribosome-inactivating protein and which cysteineis located at a position in the amino acid sequence of the analogcorresponding to position 259 in SEQ ID No: 1 or at a position in theamino acid sequence in the analog corresponding to a position from theposition corresponding to position 251 in SEQ ID NO: 1 to the carboxylterminal position of the analog.

An analog according to the present invention may be an analog ofgelonin. In an analog of gelonin according to the present invention, thecysteine may be at a position in the analog from position 244 to thecarboxyl terminal position of the analog, more preferably at a positionin the analog from position 247 to the carboxyl terminal position of theanalog, and, in these regions, most preferably at position 244, atposition 247 or at position 248 of the amino acid sequence of theanalog. It is preferred that the gelonin cysteine residues at positions44 and 50 be replaced with alanine residues.

An analog according to the present invention may be an analog of barleyribosome-inactivating protein. Preferably, a cysteine in such an analogis at a position in the analog from position 256 to the carboxylterminal position, and more preferably the cysteine is at a position inthe amino acid sequence of the analog from position 260 to the carboxylterminal position of the analog. Most preferably, in these regions, thecysteine is at position 256, at position 270 or at position 277 of theamino acid sequence of the analog.

An analog according to the present invention may be an analog ofmomordin II.

Analogs according to the present invention may have a cysteine in theamino acid sequence of the analog at a position which corresponds to aposition within one amino acid of position 259 of SEQ ID NO: 1. Such ananalog may be an analog of gelonin, of barley ribosome-inactivatingprotein, or of momordin II.

The present invention also provides a polynucleotide encoding an analogof a Type I ribosome-inactivating protein, which analog has a cysteineavailable for intermolecular disulfide bonding at an amino acid positioncorresponding to a position not naturally available for intermoleculardisulfide bonding in the Type I ribosome-inactivating protein, and whichcysteine is located at a position in the amino acid sequence of theanalog from the position corresponding to position 251 in SEQ ID NO: 1to the carboxyl terminal position of the analog. The polynucleotide mayencode an analog of gelonin, preferably an analog wherein the cysteineis at a position in the amino acid sequence of the analog from position244 to the carboxyl terminal position of the analog, more preferablywherein the cysteine is at a position in the analog from position 247 tothe carboxyl terminal position of the analog, and most preferably thecysteine is at position 244, at position 247 or at position 248 of theamino acid sequence of the analog. It is preferred that a polynucleotideaccording to the present invention encode a gelonin analog wherein thenative gelonin cysteine residues at positions 44 and 50 are replacedwith alanine residues.

A polynucleotide according to the present invention may encode an analogof barley ribosome-inactivating protein, preferably an analog whereinthe cysteine is at a position in the analog from position 256 to thecarboxyl terminal position of the analog, more preferably wherein thecysteine is at a position in the analog from position 260 to thecarboxyl terminal position of the analog, and most preferably whereinthe cysteine is at position 256, at position 270 or at position 277 ofthe amino acid sequence of the analog.

A polynucleotide according to the present invention may encode an analogof mormordin II.

The present invention provides a vector including a polynucleotideencoding an analog of a Type I ribosome-inactivating protein, whichanalog has a cysteine available for intermolecular disulfide bonding ata amino acid position corresponding to a position not naturallyavailable for intermolecular disulfide bonding in the Type Iribosome-inactivating protein and which cysteine is located at aposition in the amino acid sequence of the analog from the positioncorresponding to position 251 in SEQ ID NO: 1 to the carboxyl terminalposition of the analog.

The present invention further provides a host cell including a DNAvector encoding an analog of a Type I ribosome-inactivating protein,which analog has a cysteine available for intermolecular disulfidebonding at an amino acid position corresponding to a position notnaturally available for intermolecular disulfide bonding in the Type Iribosome-inactivating protein and which cysteine is located at aposition in the amino acid sequence of the analog from the positioncorresponding to position 251 in SEQ ID NO: 1 to the carboxyl terminalposition of the analog. In such a host cell the vector may encode ananalog of gelonin, especially an analog wherein the cysteine is atposition 247 of the amino acid sequence of the analog, such as in thehost cell deposited as ATCC Accession No. 69009.

A host cell according to the present invention may include a vectorencoding barley ribosome-inactivating protein, especially a host cellwherein the cysteine is at position 277 of the amino acid sequence ofthe analog such as in the host cell deposited as ATCC Accession No.68722.

The present invention also provides an agent toxic to a cell includingan analog of a Type I ribosome-inactivating protein linked by adisulfide bond through a cysteine to a molecule which specifically bindsto the cell, which cysteine is at an amino acid position in the analogcorresponding to a position not naturally available for intermoleculardisulfide bonding in the Type I ribosome-inactivating protein and whichcysteine is located in the amino acid sequence of the analog from theposition corresponding to position 251 in SEQ ID NO: 1 to the carboxylterminal position of the analog. The agent may include an analog ofgelonin, preferably an analog wherein the cysteine is at a position inthe analog from position 247 to the carboxyl terminal position of theanalog, and more preferably wherein the cysteine is at position 247 or248 of the amino acid sequence of analog. An agent including an analogwherein the native gelonin cysteine residues at positions 44 and 50 arereplaced with alanine residues is preferred.

An agent according to the present invention may include an analog ofbarley ribosome-inactivating protein, preferably an analog wherein thecysteine is at a position in the analog from position 260 to thecarboxyl terminal position of the analog, more preferably wherein thecysteine is at a position in the analog from position 270 to thecarboxyl terminal position of the analog, and most preferably whereinthe cysteine is at position 256, at position 270 or at position 277 ofthe amino acid sequence of the analog.

An agent according to the present invention may include an analog ofmomordin II.

The present invention provides an agent wherein the Type Iribosome-inactivating protein is linked to an antibody, particularly toan H65 antibody or to an antibody fragment, more particularly to anantibody fragment selected from the group consisting of chimeric andhuman engineered antibody fragments, and most particularly to a Fabantibody fragment, a Fab' antibody fragment or a F(ab')₂ antibodyfragment. It is highly preferred that an agent according to the presentinvention include a chimeric or human engineered antibody fragmentselected from the group consisting of a Fab antibody fragment, a Fab'antibody fragment and a F(ab')₂ antibody fragment.

A method according to the present invention for preparing an analog of aType I ribosome-inactivating protein includes the step of expressing ina suitable host cell a polynucleotide encoding a Type Iribosome-inactivating protein having a cysteine available forintermolecular disulfide bonding substituted (e.g., by site-directedmutagenesis of the natural DNA sequence encoding the RIP or by chemicalsynthesis of a DAN sequence encoding the RIP analog) at an amino acidposition corresponding to a position not naturally available forintermolecular disulfide bonding in the Type I ribosome-inactivatingprotein, which cysteine is located at a position in the amino acidsequence of the analog from the position corresponding to position 251in SEQ ID NO: 1 to the carboxyl terminal position of the analog.

A product according to the present invention may be a product of amethod including the step of expressing in a suitable host cell apolynucleotide encoding a Type I ribosome-inactivating protein having acysteine available for intermolecular disulfide bonding substituted atan amino acid position corresponding to a position not naturallyavailable for intermolecular disulfide bonding in the Type Iribosome-inactivating protein, which cysteine is located at a positionin the amino acid sequence of the analog from the position correspondingto position 251 in SEQ ID NO: 1 to the carboxyl terminal position of theanalog.

The present invention provides a method for preparing an agent toxic toa cell including the step of linking an analog of a Type Iribosome-inactivating protein through a cysteine to a molecule whichspecifically binds to the cell, which analog has the cysteine at anamino acid position corresponding to a position not naturally availablefor intermolecular disulfide bonding in the Type I ribosome-inactivatingprotein and which cysteine is located at a position in the amino acidsequence of the analog from the position corresponding to position 251in SEQ ID NO: 1 to the carboxyl terminal position of the analog.

According to the present invention, a method for treating a disease inwhich elimination of particular cells is a goal may include the step ofadministering to a patient having the disease a therapeuticallyeffective amount of an agent toxic to the cells including an analog of aType I ribosome-inactivating protein linked through a cysteine to amolecule which specifically binds to the cell, the analog having thecysteine at an amino acid position corresponding to a position notnaturally available for intermolecular disulfide bonding in the Type Iribosome-inactivating protein and the cysteine being located at aposition in the amino acid sequence of the analog from the positioncorresponding to position 251 in SEQ ID NO: 1 to the carboxyl terminalposition of the analog.

The present invention also provides an analog of a Type Iribosome-inactivating protein, wherein the analog has a cysteineavailable for intermolecular disulfide bonding located at an amino acidposition corresponding to a position not naturally available forintermolecular disulfide bonding in the Type I ribosome-inactivatingprotein and corresponding to a position on the surface of ricin A-chainin its natural conformation, and wherein the analog retainsribosome-inactivating activity of the Type I ribosome-inactivatingprotein.

Such an analog may be an analog wherein the Type I ribosome inactivatingprotein is gelonin, and is preferably an analog of gelonin wherein thecysteine is at position 10 of the amino acid sequence of the analog asencoded in a vector in a host cell deposited as ATCC Accession No.69008. Other such gelonin analogs include those wherein the cysteine isat a position 60, 103, 146, 184 or 215 in the amino acid sequence of thegelonin analog. It is preferred that the gelonin cysteine residues atpositions 44 and 50 be replaced with alanine residues in these analogs.

The present invention further provides an analog of a Type Iribosome-inactivating protein wherein the analog includes only a singlecysteine. Such an analog may be an analog of gelonin and is preferablyan analog wherein the single cysteine is at position 10, position 44,position 50 or position 247 in the amino acid sequence of the analog,but the cysteine may be located at other positions defined by theinvention as well.

The present invention provides a polynucleotide encoding an analog of aType I ribosome-inactivating protein, wherein the analog has a cysteineavailable for intermolecular disulfide bonding located at an amino acidposition corresponding to a position not naturally available forintermolecular disulfide bonding in the Type I ribosome-inactivatingprotein and corresponding to a position on the surface of ricin A-chainin its natural conformation, and wherein the analog retainsribosome-inactivating activity of the Type I ribosome-inactivatingprotein.

According to the present invention, a method for preparing an analog ofa Type I ribosome-inactivating protein may include the step ofexpressing in suitable host cell a polynucleotide encoding a Type Iribosome-inactivating protein having a cysteine available forintermolecular disulfide bonding substituted at an amino acid positioncorresponding to a position not naturally available for disulfidebonding in the Type I ribosome-inactivating protein, the cysteine islocated at a position corresponding to an amino acid position on thesurface of ricin A-chain in its natural conformation and which analogretains ribosome-inactivating activity of the Type Iribosome-inactivating protein.

The present invention provides an agent toxic to a cell including ananalog of a Type I ribosome-inactivating protein linked by a disulfidebond through a cysteine to a molecule which specifically binds to thecell, wherein the analog has a cysteine available for intermoleculardisulfide bonding located at an amino acid position corresponding to aposition not naturally available for intermolecular disulfide bonding inthe Type I ribosome-inactivating protein and corresponding to a positionon the surface of ricin A-chain in its natural conformation, and whereinthe analog retains ribosome-inactivating activity of the Type Iribosome-inactivating protein.

A method according to the present invention for preparing an agent toxicto a cell may include the step of linking an analog of a Type Iribosome-inactivating protein through a cysteine to a molecule whichspecifically binds to the cell, which analog has a cysteine availablefor intermolecular disulfide bonding located at an amino acid positioncorresponding to a position not naturally available for intermoleculardisulfide bonding in the Type I ribosome-inactivating protein andcorresponding to a position on the surface of ricin A-chain in itsnatural conformation, and which analog retains ribosome-inactivatingactivity of the Type I ribosome-inactivating protein.

A method according to the present invention for treating a disease inwhich elimination of particular cells is a goal includes the step ofadministering to a patient having the disease a therapeuticallyeffective amount of an agent toxic to the cells wherein the agentincludes an analog of a Type I ribosome-inactivating protein linked by adisulfide bond through a cysteine to a molecule which specifically bindsto the cell, which analog has a cysteine available for intermoleculardisulfide bonding located at an amino acid position corresponding to aposition not naturally available for intermolecular disulfide bonding inthe Type I ribosome-inactivating protein and corresponding to a positionon the surface of ricin A-chain in its natural conformation, and whichanalog retains ribosome-inactivating activity of the Type Iribosome-inactivating protein.

The RIP analogs are particularly suited for use as components ofcytotoxic therapeutic agents. Cytotoxic agents according to the presentinvention may be used in vivo to selectively eliminate any cell type towhich the RIP component is targeted by the specific binding capacity ofthe second component. To form cytotoxic agents RIP analogs may beconjugated to monoclonal antibodies, including chimeric and CDR-graftedantibodies, and antibody domains/fragments (e.g., Fab, Fab', F(ab')₂,single chain antibodies, and Fv or single variable domains) as well asconjugated to monoclonal antibodies genetically engineered to includefree cysteine residues are within the scope of the present invention.Examples of Fab' and F(ab')₂ fragments useful in the present inventionare described in co-pending, co-owned U.S. patent application Ser. No.07/714,175, filed Jun. 14, 1991 and in International Publication No. WO89/00999 published on Feb. 9, 1989, which are incorporated by referenceherein. RIPs according to the present invention may also be conjugatedto targeting agents other than antibodies, for example, lectins whichbind to cells having particular surface carbohydrates, or hormones,lymphokines, growth factors or other polypeptides which bindspecifically to cells having particular receptors. Immunoconjugatesincluding RIPs may be described as immunotoxins. An immunotoxin may alsoconsist of a fusion protein rather than an immunoconjugate.

The present invention provides gene fusions of an antigen-bindingportion of an antibody (e.g., an antibody light chain or truncated heavychain, or a single chain antibody) or any targeting agent listed in theforegoing paragraph, linked to a Type I RIP. The gene fusions mayinclude an RIP gene linked either at the 5' or the 3' end of an antibodygene. A DNA linker encoding a peptide segment may or may not be insertedbetween the toxin and the antibody gene. Preferably, the linker encodesa segment of the E. coli shiga-like toxin which contains two cysteineresidues participating in a disulfide bond and forming a loop thatincludes a protease sensitive amino acid sequence (e.g., SEQ ID NO: 58),or a segment of rabbit muscle aldolase which contains several potentialcathepsin cleavage sites (e.g., SEQ ID NO: 59). The Type I RIP portionof the fused genes preferably encodes gelonin, BRIP or momordin II. Alsopreferably, the antibody portion of the fused genes comprises sequencesencoding one of the chains of an antibody Fab fragment (i.e., kappa orFd) and the fused gene is co-expressed in a host cell with the other Fabgene, or the antibody portion comprises sequences encoding a singlechain antibody.

The present invention also provides a method for purifying an protein orimmunotoxin comprising a ribosome-inactivating protein and a portion ofan antibody including the steps of passing a solution containing theprotein through an anion exchange column; applying the flow-through to aprotein G column; and eluting the protein from the protein G column. Themethod may further comprise the steps of introducing the flow-through ofthe anion exchange column into a cation exchange column; exposing thecation exchange column to an eluent effective to elute said protein; andthen applying the eluted protein to a protein G column, rather thanapplying the anion exchange column flow-through directly to a protein Gcolumn.

Immunotoxins according to the present invention includingimmunoconjugates and fusion proteins are suited for treatment ofdiseases where the elimination of a particular cell type is a goal, suchas autoimmune disease, cancer and graft-versus-host disease. Theimmunotoxins are also suited for use in causing immunosuppression and intreatment of infections by viruses such as the Human ImmunodeficiencyVirus.

Specifically illustrating polynucleotide sequences according to thepresent invention are the inserts in the plasmid pING3731 in E. coliMC1061 (designated strain G274) and in the plasmid pING3803 in E. coliE104 (designated strain G275), both deposited with the American TypeCulture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md., on Oct.2, 1991, and assigned ATCC Accession Nos. 68721 and 68722, respectively.Additional polynucleotide sequences illustrating the invention are theinserts in the plasmid pING3746 in E. coli E104 (designated strain G277)and in the plasmid pING3737 in E. coli E104 (designated strain G276),which were both deposited with the ATCC on June 9, 1992, and wererespectively assigned Accession Nos. 69008 and 69009. Still otherpolynucleotide sequences illustrating the invention are the inserts inthe plasmid pING3747 in E. coli E104 (designated strain G278), in theplasmid pING3754 in E. coli E104 (designated strain G279), in theplasmid pING3758 in E. coli E104 (designated strain G280) and in theplasmid pING3759 in E. coli E104 (designated strain G281), whichplasmids were all deposited with the ATCC on Oct. 27, 1992 and wereassigned ATCC Accession Nos. 69101, 69102, 69103 and 69104,respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (RTA) (SEQ ID NO: 1) with the amino acid sequence ofthe Type I ribosome-inactivating protein gelonin (SEQ ID NO: 2), whereinstarred positions indicate amino acids invariant among the ricin A-chainand the Type I RIPs;

FIG. 2 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein BRIP (SEQ ID NO: 3), whereinstarred positions indicate amino acids invariant among the ricin A-chainand the Type I RIPs;

FIG. 3 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein momordin II (MOMOII) (SEQ ID NO:4), wherein starred positions indicate amino acids invariant among thericin A-chain and the Type I RIPs;

FIG. 4 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein luffin (SEQ ID NO: 5), whereinstarred positions indicate amino acids invariant among the ricin A-chainand the Type I RIPs;

FIG. 5 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein αtrichosanthin (TRICHO) (SEQ ID NO:6), wherein starred positions indicate amino acids invariant among thericin A-chain and the Type I RIPs;

FIG. 6 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein momordin I (MOMOI) (SEQ ID NO: 7),wherein starred positions indicate amino acids invariant among the ricinA-chain and the Type I RIPs;

FIG. 7 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein Mirabilis anti-viral protein (MAP)(SEQ ID NO: 8), wherein starred positions indicate amino acids invariantamong the ricin A-chain and the Type I RIPs;

FIG. 8 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein pokeweed antiviral protein fromseeds (PAPS) (SEQ ID NO: 9), wherein starred positions indicate aminoacids invariant among the ricin A-chain and the Type I RIPs;

FIG. 9 is a computer-generated alignment of the amino acid sequence ofthe ricin A-chain (SEQ ID NO: 1) with the amino acid sequence of theType I ribosome-inactivating protein saporin 6 (SAP6) (SEQ ID NO: 10),wherein starred positions indicate amino acids invariant among the ricinA-chain and the Type I RIPs.

DETAILED DESCRIPTION

Nucleotide sequences of genes encoding three plant Type I RIPs andexpression vectors containing the genes are provided by the presentinvention. A first plant RIP, gelonin, is produced by seeds of Geloniummultiflorum, a plant of the Euphorbiaceae family native to the tropicalforests of eastern Asia, while a second plant RIP, BRIP, is synthesizedby the common cereal grain barley. Momordin II, a third plant RIP, isproduced in Momordica balsamina seeds. Analogs of BRIP are also providedby the present invention. The analogs were genetically engineered toinclude a cysteine free to participate in a intermolecular disulfidebond and were conjugated to antibody molecules without non-specificchemical derivatization of the RIP with crosslinking agents.

Type I RIP analogs of the present invention offer distinct advantagesover the natural proteins for use as components of immunotoxins.Chemical treatment to introduce free sulfhydryl groups in the naturalproteins lacking free cysteines typically involves the non-selectivemodification of amino acid side chains. This non-selectivity oftenresults in antibodies conjugated to different sites on different RIPmolecules (i.e., a heterogeneous population of conjugates) and also in adecrease in RIP activity if antibodies are conjugated in or nearimportant regions of the RIP (e.g., the active site or regions involvedin translocation across cell membranes). In contrast, RIP analogsaccording to the present invention can be conjugated to a singleantibody through a disulfide bond to a specific residue of the analogresulting in reduced batch to batch variation of the immunoconjugatesand, in some cases, immunoconjugates with enhanced properties (e.g.,greater cytotoxicity or solubility).

Type I plant RIPs, as well as bacterial RIPs such as shiga andshiga-like toxin A-chains, are homologous to the ricin A-chain and areuseful in the present invention.

Type I RIPs may be defined and sites for substitution of a cysteine in aRIP may be identified by comparing the primary amino acid sequence ofthe RIP to the natural ricin A-chain amino acid sequence, the tertiarystructure of which has been described in Katzin et al., Proteins, 10,251-259 (1991), which is incorporated by reference herein.

Amino acid sequence alignment defines Type I RIPs in that the ricinA-chain and the Type I plant RIPs have nine invariant amino acids incommon. Based on the ricin sequence the invariant amino acids aretyrosine₂₁, arginine₂₉, tyrosine₈₀, tyrosine₁₂₃, leucine₁₄₄, glutamicacid₁₇₇, alanine₁₇₈, arginine₁₈₀, and tryptophan₂₁₁. The ricin A-chainmay be used as a model for the three-dimensional structure of Type IRIPs. A protein lacking a cysteine available for conjugation whilehaving ribosome-inactivating activity and having the nine invariantamino acids when its primary sequence is compared to the primarysequence of the ricin A-chain [according to the alignment algorithm ofMyers et al., CABIOS COMMUNICATIONS, 4(1), 11-17 (1988), implemented bythe PC/GENE program PALIGN (Intelligenetics, Inc., Mountain View,Calif.) and utilizing the Dayhoff Mutation Data Matrix (MDM-78) asdescribed in Schwartz et al., pp. 353-358 in Atlas of Protein Sequenceand Structure, 5 Supp. 3, National Biomedical Research Foundation,Washington, D.C. (1978)] is defined as a Type I RIP herein and isexpected to be useful in the present invention. "Corresponding" refersherein to amino acid positions that align when two amino acid sequencesare compared by the strategy of Myers et al., Supra.

The primary amino acid sequences of the Type I RIPs gelonin, BRIP,momordin II, luffin [see Islam et al., Agricultural Biological Chem.,54(5), 1343-1345 (199)], αtrichosanthin [see Chow et al., J. Biol.Chem., 265, 8670-8674 (1990)], momordin I [see Ho et al., BBA, 1088,311-314 (1991)], Mirabilis anti-viral protein [see Habuka et al., J.Biol. Chem., 264(12), 6629-6637 (1989)], pokeweed antiviral proteinisolated from seeds [see Kung et al., Agric. Biol. Chem., 54(12),3301-3318 (1990)] and saporin [see Benatti et al., Eur. J. Biochem.,183, 465-470 (1989)] are individually aligned with the primary sequenceof the ricin A-chain [see Halling et al., Nucleic Acids Res., 13,8019-8033 (1985)] in FIGS. 1-9, respectively, according to the algorithmof Myers et al., supra, as specified above.

FIGS. 1-9 may be utilized to predict the amino acid positions of theType I RIPs where cysteine residues may be substituted. Preferred aminoacids for cysteine substitution are on the surface of the molecule andinclude any solvent accessible amino acids that will not interfere withproper folding of the protein if replaced with a cysteine. A region ofthe ricin A-chain comprising such amino acids is the carboxyl terminalregion. Amino acids that should be avoided for replacement are thosecritical for proper protein folding, such as proline, and those that aresolvent inaccessible. Also to be avoided are the nine amino acidsinvariant among RIPs, and the amino acids in or near regions comprisingthe active site of ricin A-chain as depicted in FIG. 6 of Katzin et al.,supra.

Therefore, a preferred region of substitution for Type I RIPs is theircarboxyl terminal region which is solvent accessible and corresponds tothe carboxyl terminal region where Type II RIP A-chains and B-chains arenaturally linked by a disulfide bond. As shown in the examples, acysteine may be substituted in positions in the amino acid sequence of aType I RIP from the position corresponding to position 251 in SEQ ID NO:1 to the carboxyl terminal position of said Type I RIP, resulting in RIPanalogs which retain enzymatic activity and gain disulfide crosslinkingcapability. One preferred cysteine substitution position is near theposition which corresponds to the cysteine at position 259 in the ricinA-chain.

Immunotoxins specifically illustrating the present invention includingcytotoxic agents and gene fusion products are particularly suited foruse in treatment of human autoimmune disease where T-cell function isimplicated. Treatment of autoimmune diseases with immunotoxins isdescribed in co-owned U.S. patent application Ser. No. 07/306,433 filedon Sep. 13, 1991 and in International Publication No. WO89/06968published Aug. 10, 1989, which are incorporated by reference herein.Examples of autoimmune diseases are systemic lupus erythematosus,scleroderma diseases (including lichen sclerosus, morphea and lichenplanus), rheumatoid arthritis, chronic thyroiditis, pemphigus vulgaris,diabetes mellitus type 1, progressive systemic sclerosis, aplasticanemia, myasthenia gravis, myositis, Sjogrens disease, Crohn's disease,ulcerative colitis, and primary biliary cirrhosis. Autoimmunity is alsoimplicated in multiple sclerosis, uveitis, psoriasis and Meniere'sdisease. A general description of various autoimmune diseases may befound in Rose and Mackey, Eds., The Autoimmune Diseases, Academic Press(1985).

The immunotoxins may be administered to a patient either singly or in acocktail containing two or more immunotoxins, other therapeutic agents,compositions, or the like, including, but not limited to,immunosuppressive agents, tolerance-inducing agents, potentiators andside-effect relieving agents. Particularly preferred areimmunosuppressive agents useful in suppressing allergic reactions of ahost. Preferred immunosuppressive agents include prednisone,prednisolone, DECADRON (Merck, Sharp & Dohme, West Point, Pa.),cyclophosphamide, cyclosporine, 6-mercaptopurine, methotrexate,azathioprine and i.v. gamma globulin or their combination. Preferredpotentiators include monensin, ammonium chloride, perhexiline,verapamil, amantadine and chloroquine. All of these agents areadministered in generally-accepted efficacious dose ranges such as thosedisclosed in the Physician's Desk Reference, 41st Ed., Publisher EdwardR. Barnhart, N.J. (1987). Patent Cooperation Treaty (PCT) patentapplication WO 89/069767 published on Aug. 10, 1989, disclosesadministration of an immunotoxin as an immunosuppressive agent and isincorporated by reference herein.

Anti-T cell immunotoxins may be formulated into either an injectable ortopical preparation. Parenteral formulations are known and are suitablefor use in the invention, preferably for intramuscular or intravenousadministration. The formulations containing therapeutically-effectiveamounts of anti-T cell immunotoxins are either sterile liquid solutions,liquid suspensions or lyophilized versions, and optionally containstabilizers or excipients. Lyophilized compositions are reconstitutedwith suitable diluents, e.g., water for injection, saline, 0.3% glycineand the like, at a level of about from 0.01 mg/kg of host body weight to10 mg/kg where the biological activity is less than or equal to 20 ng/mlwhen measured in a reticulocyte lysate assay. Typically, thepharmaceutical compositions containing anti-T cell immunotoxins will beadministered in a therapeutically effective dose in a range of fromabout 0.01 mg/kg to about 5 mg/kg of the patient. A preferred,therapeutically effective dose of the pharmaceutical compositioncontaining anti-T cell immunotoxin will be in a range of from about 0.01mg/kg to about 0.5 mg/kg body weight of the patient administered overseveral days to two weeks by daily intravenous infusion, each given overa one hour period, in a sequential patient dose-escalation regimen.

Anti-T cell immunotoxin is formulated into topical preparations forlocal therapy by including a therapeutically effective concentration ofanti-T cell immunotoxin in a dermatological vehicle. The amount ofanti-T cell immunotoxin to be administered, and the anti-T cellimmunotoxin concentration in the topical formulations, depends upon thevehicle selected, the clinical condition of the patient, the systemictoxicity and the stability of the anti-T cell immunotoxin in theformulation. Thus, a physician knows to employ the appropriatepreparation containing the appropriate concentration of anti-T cellimmunotoxin in the formulation, as well as the appropriate amount offormulation to administer depending upon clinical experience with thepatient in question or with similar patents. The concentration of anti-Tcell immunotoxin for topical formulations is in the range of greaterthan from about 0.1 mg/ml to about 25 mg/ml. Typically, theconcentration of anti-T cell immunotoxin for topical formulations is inthe range of greater than from about 1 mg/ml to about 20 mg/ml. Soliddispersions of anti-T cell immunotoxin as well as solubilizedpreparations can be used. Thus, the precise concentration to be used inthe vehicle is subject to modest experimental manipulation in order tooptimize the therapeutic response. Greater than about 10 mg anti-T cellimmunotoxin/100 grams of vehicle may be useful with 1% w/w hydrogelvehicles in the treatment of skin inflammation. Suitable vehicles, inaddition to gels, are oil-in-water or water-in-oil emulsions usingmineral oils, petroleum and the like.

Anti-T cell immunotoxin is optionally administered topically by the useof a transdermal therapeutic system [Barry, Dermatological Formulations,p. 181 (1983) and literature cited therein]. While such topical deliverysystems have been designed for transdermal administration of lowmolecular weight drugs, they are capable of percutaneous delivery. Theymay be readily adapted to administration of anti-T cell immunotoxin orderivatives thereof and associated therapeutic proteins by appropriateselection of the rate-controlling microporous membrane.

Topical preparations of anti-T cell immunotoxin either for systemic orlocal delivery may be employed and may contain excipients as describedabove for parenteral administration and other excipients used in atopical preparation such as cosolvents, surfactants, oils, humectants,emollients, preservatives, stabilizers and antioxidants. Anypharmacologically-acceptable buffer may be used, e.g., Tris or phosphatebuffers. The topical formulations may also optionally include one ormore agents variously termed enhancers, surfactants, accelerants,adsorption promoters or penetration enhancers, such as an agent forenhancing percutaneous penetration of the anti-T cell immunotoxin orother agents. Such agents should desirably possess some or all of thefollowing features as would be known to the ordinarily skilled artisan:pharmacological inertness, non-promotive of body fluid or electrolyteloss, compatible with anti-T cell immunotoxin (non-inactivating), andcapable of formulation into creams, gels or other topical deliverysystems as desired.

Anti-T cell immunotoxin may also be administered by aerosol to achievelocalized delivery to the lungs. This is accomplished by preparing anaqueous aerosol, liposomal preparation or solid particles containingimmunotoxin. Ordinarily, an aqueous aerosol is made by formulating anaqueous solution or suspension of anti-T cell immunotoxin together withconventional pharmaceutically acceptable carriers and stabilizers. Thecarriers and stabilizers vary depending upon the requirements for theparticular anti-T cell immunotoxin, but typically include: nonionicsurfactants (Tweens, Pluronics, or polyethylene glycol); innocuousproteins like serum albumin, sorbitan esters, oleic acid, lecithin;amino acids such as glycine; and buffers, salts, sugars or sugaralcohols. The formulations may also include mucolytic agents as well asbronchodilating agents. The formulations are sterile. Aerosols generallyare prepared from isotonic solutions. The particles optionally includenormal lung surfactants.

Aerosols may be formed of the particles in aqueous or nonaqueous (e.g.,fluorocarbon propellant) suspension. Such particles include, forexample, intramolecular aggregates of anti-T cell immunotoxin orderivatives thereof or liposomal or microcapsular-entrapped anti-T cellimmunotoxin or derivatives thereof. The aerosols should be free of lungirritants, i.e., substances which cause acute bronchoconstriction,coughing, pulmonary edema or tissue destruction. However, nonirritatingabsorption-enhancing agents are suitable for use herein. Sonicnebulizers are preferably used in preparing aerosols. Sonic nebulizersminimize exposing the anti-T cell immunotoxin or derivatives thereof toshear, which can result in degradation of anti-T cell immunotoxin.

Anti-T cell immunotoxin may be administered systemically, rather thantopically, by injection intramuscularly, subcutaneously, intrathecallyor intraperitoneally or into vascular spaces, particularly into thejoints, e.g., intraarticular injection at a dosage of greater than about1 μg/cc joint fluid/day. The dose will be dependent upon the propertiesof the anti-T cell immunotoxin employed, e.g., its activity andbiological half-life, the concentration of anti-T cell immunotoxin inthe formulation, the site and rate of dosage, the clinical tolerance ofthe patient involved, the autoimmune disease afflicting the patient andthe like, as is well within the skill of the physician.

The anti-T cell immunotoxins of the present invention may beadministered in solution. The pH of the solution should be in the rangeof pH 5 to 9.5, preferably pH 6.5 to 7.5. The anti-T cell immunotoxin orderivatives thereof should be in a solution having a suitablepharmaceutically-acceptable buffer such as phosphate,Tris(hydroxymethyl)aminomethane-HCl or citrate and the like. Bufferconcentrations should be in the range of 1 to 100 mM. The solution ofanti-T cell immunotoxin may also contain a salt, such as sodium chlorideor potassium chloride in a concentration of 50 to 150 mM. An effectiveamount of a stabilizing agent such as an albumin, a globulin, a gelatin,a protamine or a salt of protamine may also be included, and may beadded to a solution containing anti-T cell immunotoxin or to thecomposition from which the solution is prepared.

Systemic administration of anti-T cell immunotoxin is made daily and isgenerally by intramuscular injection, although intravascular infusion isacceptable. Administration may also be intranasal or by othernonparenteral routes. Anti-T cell immunotoxin may also be administeredvia microspheres, liposomes or other microparticulate delivery systemsplaced in certain tissues including blood. Topical preparations areapplied daily directly to the skin or mucosa and are then preferablyoccluded, i.e., protected by overlaying a bandage, polyolefin film orother barrier impermeable to the topical preparation.

The following examples illustrate practice of the invention but are notto be construed as limiting the invention. Example 1 is a description ofthe cloning of a cDNA encoding the Type I RIP gelonin. Example 2describes the construction of recombinant expression vectors containingthe gelonin gene. Described in Example 3 are constructions of variousanalogs of gelonin having a single cysteine available for disulfidebonding. Example 4 describes the testing of recombinant gelonin and thegelonin analogs for the capacity to inhibit protein synthesis in areticulocyte lysate assay. Example 5 presents descriptions of thepreparation of various gelonin immunoconjugates. Example 6 describes ofthe testing of the immunoconjugates for the capacity to act as cytotoxicagents in a whole cell kill assay. Example 7 presents solubility andstability characteristics of the immunoconjugates. Examples 8 presentsresults of in vivo pharmacokinetic and immunogenicity studies of thegelonin immunoconjugates and Example 9 presents results of testing ofthe immunoconjugates for the capacity to deplete human T cells in ahuman peripheral blood lymphocyte-reconstituted, severe combinedimmunodeficient mouse model. Described in Example 10 are various genefusions of gelonin DNA sequences and sequences encoding antibodyfragments. Expression of products of the gene fusions products andtesting of the products in the reticulocyte and whole cell kill assaysare described in Example 11. Example 12 is a description of theconstruction of gelonin gene fusions to single chain antibodies. Example13 describes the cloning of a cDNA encoding the Type I RIP BRIP,construction of expression vectors containing the BRIP gene, productionof BRIP analogs having a single cysteine available for disulfidebonding, testing of the analogs in the reticulocyte lysate assay, andconstruction of the BRIP immunoconjugates and testing of their activityin the whole cell kill assay. Example 14 describes the cloning of a cDNAencoding momordin II and construction of expression vectors containingthe momordin II gene.

Example 1

The cloning of the gelonin gene according to the present inventionobviates the requirement of purifying the RIP gene product from itsrelatively scarce natural source, G. multiflorum seeds, and allowsdevelopment of gelonin analogs conjugatable to antibodies without priorchemical derivatization and development of gelonin gene fusion products.One formidable hurdle in the cloning of the gene was that the availableGelonium seeds are old and inviable, making preparation of intactmessenger RNA from the seeds impossible. Cloning the gene from cDNAprepared from messenger RNA was thus impractical and total RNA wasutilized to generate cDNA. Using total RNA to make cDNA under normalcircumstances, i.e., when mRNA may be utilized, is not desirable becausetotal RNA typically comprises about 95% ribosomal RNA.

Preparation of RNA from G. multiflorum Seeds

Total. RNA was prepared from Gelonium seeds (Dr. Michael Rosenblum, M.D.Anderson Cancer Center, Houston, Tex.) by a modification of theprocedure for preparation of plant RNA described in Ausubel et al.,eds., Current Protocols in Molecular Biology:, Wiley & Sons, 1989.Briefly, 4.0 grams of seeds were ground to a fine powder in a pre-cooled(-70° C.) mortar and pestle with liquid N₂. The powder was added to 25ml Grinding buffer (0.18M Tris, 0.09M LiC1, 4.5mM EDTA, 1% SDS, pH 8.2)along with 8.5 ml of phenol equilibrated with TLE (0.2M Tris, 0.1M LiC1,5mM EDTA pH8.2). The mixture was homogenized using a Polytron PT-1035(#5 setting). 8.5 ml of chloroform was added, mixed and incubated at 50°C. for 20 minutes. The mixture was centrifuged at 3K for 20 minutes in arotor precooled to 4° C. and the aqueous phase was transferred to a newtube. 8.5 ml of phenol was added followed by 8.5 ml of chloroform andthe mixture was recentrifuged. This extraction was repeated 3 times. TheRNA in the aqueous phase was then precipitated by adding 1/3 volume 8MLiC1, and incubated at 4 ° C. for 16 hours. Next, the RNA was pelletedby centrifugation for 20 minutes at 4 ° C. The pellet was washed with 5ml of 2M LiC1, recentrifuged and resuspended in 2 ml of water. The RNAwas precipitated by addition of NaOAc to 0.3M and 2 volumes of ethanol.The RNA was stored in 70% ethanol at -70° C.

cDNA Preparation

cDNA was prepared from total Gelonium RNA by two similar methods. Thefirst method involved making a cDNA library in the bacterial expressionplasmid pcDNAII using the Librarian II cDNA Library Construction Systemkit (Invitrogen). About 5 μg of total RNA was converted to first strandcDNA with a 1:1 mixture of random primers and oligo-dT. Second strandsynthesis with DNA polymerase I was performed as described by the systemmanufacturer. Double stranded cDNA was ligated to BstX1 linkers and sizefractionated. Pieces larger than about 500 bp were ligated into theexpression vector provided in the kit. Individual vectors wereintroduced into E. coli either by transformation into high-efficiencycompetent cells or by electroporation into electrocompetent cells.Electroporation was performed with a BTX100 unit (BTX, San Diego, CA) in0.56 μ Flatpack cells as recommended by BTX based on the method of Doweret al., Nucleic Acids Res., 16, 6127-6145 (1988), at a voltage amplitudeof 850 V and a pulse length of 5 mS. The resulting library consisted ofapproximately 150,000 colonies.

The second method involved generating cDNA using the RNA-PCR kit sold byPerkin-Elmer-Cetus. About 100 ng of total Gelonium RNA was used astemplate for cDNA synthesis.

Determination of the Gelonin Protein Sequence

The partial sequence of the native gelonin protein was determined bydirect amino acid sequence analysis by automated Edman degradation asrecommended by the manufacturer using an Applied Biosystems model 470Aprotein sequencer. Proteolytic peptide fragments of gelonin (isolatedfrom the same batch of seeds as the total RNA) were sequenced.

Cloning of the Gelonin Gene

Three overlapping gelonin cDNA fragments were cloned and a compositegelonin gene was assembled from the three fragments.

(1) Cloning of the Fragment Encoding the Middle Amino Acids of Geloninin Vector pING3823.

Degenerate DNA primers based on the gelonin partial amino acid sequenceswere used to amplify by PCR, segments of the cDNA generated with thePerkin-Elmer-Cetus kit. Six primers were designed based on regions ofthe gelonin amino acid sequence where degeneracy of the primers could beminimized. Appropriate pairs of primers were tested for amplification ofa gelonin gene fragment. When products of the expected DNA size wereidentified as ethidium bromide-stained DNA bands on agarose gels, theDNA was treated with T4 DNA polymerase and then purified from an agarosegel. Only the primer pair consisting of primers designated gelo-7 andgelo-5 yielded a relatively pure product of the expected size. Thesequences of degenerate primers gelo-7 and gelo-5 are set out belowusing IUPAC nucleotide symbols.

    ______________________________________                                        Gelo-7 (SEQ ID NO: 14)                                                        5' TTYAARGAYGCNCCNGAYGCNGCNTAYGARGG 3'                                        Gelo-5 (SEQ ID NO: 15)                                                        3' TTYTTYATRATRCANTGNCGNCANCTRGTYCA 5'                                        ______________________________________                                    

Primer gelo-7 corresponds to amino acids 87-97 of gelonin while primergelo-5 corresponds to amino acids 226-236. The blunt-ended DNA fragment(corresponding to amino acids 87 to 236 of gelonin) generated withprimers gelo-7 and gelo-5 was cloned into pUC 18 (BRL, Gaithersburg,Maryland). The DNA sequence of the insert was determined, and thededuced amino acid sequence based on the resulting DNA sequence matchedthe experimentally determined gelonin amino acid sequence. The clonecontaining this gelonin segment is denoted pING3726.

The insert of clone pING3726 was labeled with ³² P and used as a probeto screen the 150,000-member Gelonium cDNA library. Only one clonehybridized to the library plated in duplicate. This clone was purifiedfrom the library and its DNA sequence was determined. The clone containsa fragment encoding 185 of the 270 amino acids of gelonin (residues25-209) and is denoted pING3823.

(2) Cloning of the Fragment Encoding the N-terminal Amino Acids ofGelonin.

Based on the sequence determined for the gelonin gene segment inpING3726, exact oligonucleotide primers were designed as PCRamplification primers to be used in conjunction with a degenerate primerto amplify a 5' gelonin gene fragment and with a nonspecific primer toamplify a 3' gelonin gene fragment. cDNA generated using thePerkin-Elmer-Cetus RNA-PCR kit was amplified.

To amplify the 5'-end of the gelonin gene, PCR amplification with adegenerate primer gelo-1 and an exact primer gelo-10 was performed. Thesequences of the primers are set out below.

    ______________________________________                                        Gelo-1 (SEQ ID NO: 16)                                                        5' GGNYTNGAYACNGTNWSNTTYWSNACNAARGG 3'                                        Gelo-10 (SEQ ID NO: 17)                                                       3' TGTCTGAACCCGTAACTTGGTAA 5'                                                 ______________________________________                                    

Primer gelo- 1 corresponds to amino acids 1-11 of the gelonin gene whileprimer gelo-10 corresponds to amino acids 126-133. The product from thereaction was reamplified with gelo-1 (SEQ ID NO: 16) and gelo-11 (anexact primer comprising sequences encoding amino acids 119-125 ofgelonin) to confer specificity to the reaction product. The sequence ofprimer gelo-11 is listed below.

    ______________________________________                                        Gelo- II (SEQ ID NO: 18)                                                      3' CACTCTTCCGTATATCTCTCTGT 5'                                                 ______________________________________                                    

Hybridization with an internal probe confirmed that the desired specificgelonin DNA fragment was amplified. This fragment was cloned into pUC18,and the vector generated was designated pING3727. The fragment wassequenced revealing that the region of the fragment (the first 27nucleotides) corresponding to part of the degenerate primer gelo-1 couldnot be translated to yield the amino acid sequence upon which primergelo-1 was originally based. This was not unexpected considering thedegeneracy of the primer. The fragment was reamplified from the GeloniumcDNA with exact primers gelo-11 (SEQ ID NO: 18) and gelo-5' (whichextends upstream of the 5' end of the gelonin gene in addition toencoding the first 16 amino acids of gelonin). The sequence of primergelo-5' is set out below.

    ______________________________________                                        Gelo-5'(SEQ ID NO: 19)                                                        5' TCAACCCGGGCTAGATACCGTGTCAT                                                 TCTCAACCAAAGGTGCCACTTATATTA 3'                                                ______________________________________                                    

The resulting DNA fragment encodes the first 125 amino acids of gelonin.While the majority of the sequence is identical to the natural geloningene, the first 32 nucleotides of the DNA fragment may not be. For thepurposes of this application this N-terminal fragment is referred to asfragment GELl-125.

(3) Cloning of the Fragment Encoding the C-terminal Amino Acids ofGelonin.

To amplify the 3'-end of the gelonin gene as well as 3'untranslatedsequences, PCR amplification with exact primers gelo-9 and XE-dT wasperformed. The sequence of each of the primers is set out below.

    ______________________________________                                        Gelo-9 (SEQ ID NO: 20)                                                        5' CTTCATTTTGGCGGCACGTATCC 3'                                                 XE-DT (SEQ ID NO: 21)                                                         3' TTTTTTTTTTTTTTTTTTTTTAG                                                    GGTGCATTCGAACGTCGGAGCTC 5'                                                    ______________________________________                                    

Primer gelo-9 corresponds to amino acids 107-113 of gelonin. PrimerXE-dT consists of an 3' oligo-dT portion and a 5' portion containing therestriction sites HindlII and XhoI, and will prime any poly A-containingcDNA. The reaction product was reamplified with exact primers gelo-8 andXE. The sequences of primers gelo-8 and XE are set out below.

    ______________________________________                                        Gelo-8 (SEQ ID NO: 22)                                                        5' CTCGCTGGAAGGTGAGAA 3'                                                      XE (SEQ ID NO: 23)                                                            3' AGGGTGCATTCGAACGTCGGAGCTC 5'                                               ______________________________________                                    

Primer gelo-8 consists of sequences encoding amino acids 115-120 ofgelonin while the primer XE corresponds to the 5' portion of the XE-dTprimer which contains HindlII and XhoI restriction sites. Hybridizationwith internal probes confirmed that the desired gelonin gene fragmentwas amplified. The fragment was cloned into pUC 18 by two differentmethods. First, it was cloned as a blunt-ended fragment into the SmaIsite of pUC 18 (the resulting vector was designated pING3728) and,second, it was cloned as an EcoRI to HindlII fragment into pUC18 (thisvector was designated pING3729). Both vector inserts were sequenced. Theinsert of pING3728 encodes amino acids 114-270 of gelonin, while theinsert of pING3729 encodes amino acids 184-270 of gelonin plus other 3'sequences.

(4) Assembly of the overlapping gelonin DNA fragments into a compositegelonin gene

To reassemble the C-terminal two-thirds of the gelonin gene, vectorpING3729 was cut with SspI (one SspI site is located within the vectorand the second is located about 80 bp downstream of the terminationcodon of the insert in the vector) and an XhoI linker (8 bp, New EnglandBiolabs) was ligated to the resulting free ends. The DNA was then cutwith XhoI and EcoRI, and the 350 bp fragment generated, encoding aminoacids 185-270 of gelonin, was isolated. This 350 bp fragment was ligatedadjacent to a NcoI to EcoRI fragment from pING3823 encoding amino acids37-185 of gelonin in a intermediate vector denoted pING3730, thusreassembling the terminal 87% of the gelonin gene (amino acids 37-270).

Next, fragment GEL1-125 was cut with SmaI and NcoI, resulting in afragment encoding amino acids 1-36 of gelonin which was ligated alongwith the NcoI to XhoI fragment of pING3730 into the vector pIC100.[pIC100 is identical to pING1500 described in Better et al., Science,240, 1041-1043 (1988), except that it lacks 37 bp upstream of the pelBleader sequence. The 37 bp were eliminated by digestion of pING1500 withSphI and EcoRI, treatment with T4 polymerase and religation of thevector. This manipulation regenerated an EcoRI site in the vector whileeliminating other undesirable restriction sites.] Before ligation, thevector pIC100 had previously been digested with SstI, treated with T4polymerase, and cut with XhoI. The ligation generated a new vectorcontaining a complete gelonin gene that was designated plasmid pING3731(ATCC Accession No. 68721 ). The complete DNA sequence of the geloningene is set out in SEQ ID NO: 11.

Example 2 Construction of Expression Vectors Containing the Gelonin Gene

A first E. coli expression vector was constructed containing the geloningene linked to the Erwinia carotovora pelB leader sequence, and to theSalmonella typhimurium araB promoter. A basic vector containing the araBpromoter is described in co-owned U.S. Pat. No. 5,028,530 issued Jul. 2,1991 which is incorporated by reference herein. The vector containingthe arab promoter was cut with EcoRI and XhoI. Two DNA fragments werethen ligated in tandem immediately downstream of the promoter. Thefragment ligated adjacent to the promoter was a 131 bp fragment derivedfrom SstI digestion, T4 polymerase treatment and digestion with EcoRI ofthe plC100 vector which includes the leader sequence of the E.carotovora pelB gene. The translated leader sequence is a signal forsecretion of the respective protein through the cytoplasmic membrane.The fragment ligated downstream of the leader sequence was a Smai toXhoI fragment from pING3731 which contains the complete gelonin gene.Thus, the expression vector contains the gelonin gene linked to the pelBleader sequence and the araB promoter. This plasmid is designatedpING3733.

A second expression vector may be constructed that is identical to thefirst except that the gelonin gene sequences encoding the nineteenC-terminal amino acids of gelonin are not included. The cDNA sequence ofthe gelonin gene predicted a 19 residue C-terminal segment that was notdetected in any peptide fragments generated for determination of thegelonin amino acid sequence. These 19 amino acids may represent apeptide segment that is cleaved from the mature toxinpost-translationally, i.e. that is not present in the native protein. Asimilar C-terminal amino acid segment was identified in the plant toxinαtrichosanthin [Chow et al., J Biol. Chem., 265, 8670-8674 (1990)].Therefore, the expression product without the C-terminal fragment may beof importance.

For construction of a gelonin expression vector without the 19C-terminal amino acids of gelonin, PCR was used to amplify and alter the3'l -end of the gene. pING3728 was amplified with primers gelo-14 andgelo-9 (SEQ ID NO: 20). The sequence of primer gelo-14 is set out below.

    ______________________________________                                        Gelo-14 (SEQ ID NO: 24)                                                        ##STR1##                                                                     ______________________________________                                    

Primer gelo-14, which corresponds to gelonin amino acids 245-256,introduces a termination codon (underlined in the primer sequence) inthe gelonin gene sequence which stops transcription of the gene beforethe sequences encoding the terminal 19 amino acids of the gelonin andalso introduces a XhoI site immediately downstream of the terminationcodon. The PCR product was cut with XhoI and EcoRI, and the resulting208 bp fragment encoding amino acids 185-251 of gelonin was purifiedfrom an agarose gel. This fragment was ligated adjacent to the NcoI toEcoRI fragment from pING3823 encoding amino acids 37-185 of gelonin togenerate plasmid pING3732. A final expression vector, pING3734,containing a gelonin gene with an altered 3'-end was generated bysubstituting an NcoI to XhoI fragment encoding amino acids 37-251 ofgelonin from pING3732 into pING3733.

Identification of the Native Gelonin 5'-End

Inverse PCR was used to identify a cDNA clone encoding the 5'-end of themature gelonin gene. 5 μg of total G. multiflorum RNA was converted tocDNA using the Superscript Plasmid System (BRL, Gaithersburg, Maryland)with Gelo-11 (SEQ ID NO: 18) as a primer. Gelonin cDNA was self-ligatedto generate covalent circular DNA and the ligated DNA was amplified byPCR with oligonucleotides Gelo-9 (SEQ ID NO: 20) and Gelo-16. Thesequence of primer Gel-16 is set out below.

    ______________________________________                                        Gelo-16 (SEQ ID NO: 25)                                                       5' GTAAGCAGCATCTGGAGCATCT 3'                                                  ______________________________________                                    

The PCR product was size-fractionated on an agarose gel and DNAs largerthan 300 bp were cloned into SmaI cut pUC18. Several clones weresequenced with the primer Gelo-18, the sequence of which is set outbelow.

    ______________________________________                                        Gelo-18 (SEQ ID NO: 26)                                                       5' CATTCAAGAAATTCACGTAGG 3'                                                   ______________________________________                                    

A clone identified as having the largest gelonin-specific insert wasdesignated pING3826. The DNA sequence of pING3826 included the first 32nucleotides of the natural, mature gelonin gene not necessarily presentin gelonin expression plasmids pING3733 and pING3734. The complete DNAsequence of the natural gelonin gene is set out in SEQ ID NO: 57.

Construction of Expression Vectors Containing a Gelonin Gene with aNatural 5'End

Derivatives of expression vectors pING3733 and pING3734 (describedabove) containing a gelonin gene with the natural 5' sequence weregenerated as follows. The 5'-end of gelonin was amplified from pING3826with the PCR primers Gelo-16 (SEQ ID NO: 24) and Gelo-17, the sequenceof which is set out below.

Gelo-17 (SEQ ID NO: 27)

5'GGCCTGGACACCGTGAGCTTTAG 3'

The 285 bp PCR product was treated with T4 polymerase and cut with NcoI.The resulting 100 bp 5'-end DNA fragment was isolated from an agarosegel and ligated adjacent to the 120 bp pelB leader fragment from plC100(cut with SstI, treated with T4 polymerase and cut with PstI) intoeither pING3733 or pING3734 digested with PstI and NcoI. The resultingplasmids pING3824 and pING3825 contain the entire native gelonin geneand the native gelonin gene minus the nineteen amino acid carboxylextension, respectively, linked to the pelB leader and under thetranscriptional control of the araB promoter. The gene construct withoutthe nineteen amino acid carboxyl extension in both pING3734 and pING3825encodes a protein product referred to in this application as"recombinant gelonin."

Purification of Recombinant Gelonin

Recombinant gelonin was purified by the following procedure: E. colifermentation broth was concentrated and buffer-exchanged to 10 mM sodiumphosphate at pH 7.0 by using an S10Y10 cartridge over a DC10 unit(Areicon) the concentrated and buffer-exchanged material was applied toa CM52 column (100 g, 5×10 cm). The column was washed with 1 L ofstarting buffer and eluted with a 0 to 300 mM NaCl gradient in startingbuffer (750 ml total volume). The pure gelonin containing fractions werepooled (elution was from 100-250 mM NaCl), concentrated over an AmiconYM10 membrane, equilibrated with 10 mM sodium phosphate buffer, pH 7.0,and stored frozen at -20°C. A further purification step was attemptedusing Blue Toyopearl chromatography. However, this procedure did notresult in an increased purity of material, and resulted in anapproximate 50% loss of the starting material.

Example 3 Assembly of gelonin genes with cysteine residues available forconjugation

The gelonin protein has two cysteine residues at positions 44 and 50which are linked by an endogenous disulfide bond. The protein containsno free cysteine residue directly available for conjugation toantibodies or other proteins. Analogs of gelonin which contain a freecysteine residue available for conjugation were generated by threedifferent approaches. In one approach, various residues along theprimary sequence of the gelonin were replaced with a cysteine residue,creating a series of analogs which contain an odd number of cysteineresidues. In another approach, one of the two endogenous cysteines wasreplaced by alanine, creating a molecule which lacks an intrachaindisulfide bond but contains a single, unpaired cysteine. In yet anotherapproach both endogenous cysteines were replaced by alanines and a thirdnon-cysteine residue was replaced by a cysteine, creating an analog witha single, unpaired cysteine.

Fifteen analogs of gelonin were constructed. Ten non-cysteine residuesof gelonin were targeted for substitution with a cysteine residue.Comparison of the amino acid sequence of gelonin to the natural aminoacid sequence and tertiary structure of the ricin A-chain (see FIG. 1)suggested that these positions would be at the surface of the moleculeand available for conjugation. Each of the ten gelonin analogs include acysteine substituted in place of one of the following residues:lysine₁₀, asparagine₆₀, isoleucine103, aspartic acid₁₄₆, arginine₁₈₄,serine₂₁₅, asparagine₂₃₉, lysine₂₄₄, aspartic acid₂₄₇, and lysine₂₄₈,and the analogs have respectively been designated Gel_(c10), Gel_(c60),Gel_(c103), Gel_(c146), Gel_(c184), Gel_(c215), Gel_(c239), Gel_(c244),Gel_(c247), and Gel_(c248).

Two analogs of gelonin were constructed in which one of the nativegelonin cysteines that participates in an endogenous disulfide bond wasreplaced with a non-cysteine residue. Specifically, the cysteine atposition 50 was replaced with an alanine residue, creating a geloninanalog (designated Gel_(c44)) which has a cysteine available fordisulfide bonding at position 44. Conversely, the cysteine at position44 was replaced with an alanine residue, resulting in an analog(designated Gel_(c50)) which has a cysteine available for disulfidebonding at position 50. The combined series of the foregoing twelveanalogs thus spans the entire length of the mature gelonin protein.

Another gelonin analog (Gel_(c44AC50A)) was constructed in which bothnative gelonin cysteines were replaced with alanines. Two additionalanalogs were constructed that have alanine residues substituted in placeof both native cysteines and have either a cysteine residue substitutedin place of the native lysine at position 10 (GelC10_(c44AC50A)) or acysteine residue substituted in place of the native aspartate atposition 247 (GelC247_(c44AC50A)).

The variants of recombinant gelonin were constructed by restrictionfragment manipulation or by overlap extension PCR with syntheticoligonucleotides. The sequences of the primers used for PCR are set outbelow. In each mutagenic primer sequence, the nucleotides correspondingto the changed amino acid, either a cysteine or an alanine residue, areunderlined.

    ______________________________________                                        Gelo-9 (SEQ ID NO: 20)                                                        Gelo-11 (SEQ ID NO: 18)                                                       Gelo-16 (SEQ ID NO: 25)                                                       Gelo-17 (SEQ ID NO: 27)                                                       Gelo-18 (SEQ ID NO: 26)                                                       Gelo-19 (SEQ ID NO: 60)                                                       5' CAGCCATGGAATCCCATRGCTG 3'                                                  GeloC-1 (SEQ ID NO: 28)                                                        ##STR2##                                                                     GeloC-2 (SEQ ID NO: 29)                                                        ##STR3##                                                                     GeloC-3-2 (SEQ ID NO: 30)                                                      ##STR4##                                                                     GeloC-4 (SEQ ID NO: 31)                                                        ##STR5##                                                                     GeloC-5 (SEQ ID NO: 32)                                                        ##STR6##                                                                     GeloC-6 (SEQ ID NO: 33)                                                        ##STR7##                                                                     GeloC-9 (SEQ ID NO: 34)                                                        ##STR8##                                                                     GeloC-10 (SEQ ID NO: 35)                                                       ##STR9##                                                                     GeloC-13 (SEQ ID NO: 36)                                                       ##STR10##                                                                    GeloC-14 (SEQ ID NO: 37)                                                       ##STR11##                                                                    GeloC-15 (SEQ ID NO: 38)                                                       ##STR12##                                                                    GeloC-16 (SEQ ID NO: 39)                                                       ##STR13##                                                                    GeloC-17 (SEQ ID NO: 40)                                                       ##STR14##                                                                    GeloC-18 (SEQ ID NO: 41)                                                       ##STR15##                                                                    TTTTTTGCGCAGCAATGGG 3'                                                        GeloC-19 (SEQ ID NO: 42)                                                       ##STR16##                                                                    GeloC-20 (SEQ ID NO: 61)                                                       ##STR17##                                                                    GeloC-21 (SEQ ID NO: 62)                                                       ##STR18##                                                                    GeloC-22 (SEQ ID NO: 63)                                                       ##STR19##                                                                    GeloC-23 (SEQ ID NO: 64)                                                       ##STR20##                                                                    CATTTTCATCAAGTTTCTTG 3'                                                       GeloC-24 (SEQ ID NO: 65)                                                       ##STR21##                                                                    GeloC-25 (SEQ ID NO: 66)                                                       ##STR22##                                                                    GeloC-26 (SEQ ID NO: 67)                                                       ##STR23##                                                                    GeloC-27 (SEQ ID NO: 68)                                                       ##STR24##                                                                    GeloC-28 (SEQ ID NO: 69)                                                       ##STR25##                                                                    GeloC-29 (SEQ ID NO: 70)                                                       ##STR26##                                                                    ara B2 (SEQ ID NO: 43)                                                        5' GCGACTCTCTACTGTITC 3'                                                      HINDIII-2 (SEQ ID NO: 44)                                                     5' CGTTAGCAATTTAACTGTGAT 3'                                                   ______________________________________                                    

(1) Specifically, a cysteine was introduced at amino acid 247 of gelonin(an aspartic acid which corresponds to the cysteine at position 259 inthe ricin A-chain) by PCR with mutagenic primers GeloC-3-2 and GeloC-4in conjunction with primers HINDIII-2 (a primer located in the vectorportion of pING3734 or pING3825), Gelo-9 and Gelo-8. Template DNA(pING3734) was amplified with GeloC-3-2 and HINDIII-2 and in aconcurrent reaction with GeloC-4 and Gelo-9. The products of thesereactions were mixed and amplified with the outside primers Gelo-8 andHINDIII-2. The reaction product was cut with EcoRI and XhoI, purified,and was inserted into plasmid pING3825 in a three-piece ligation. TheDNA sequence of the Gel_(c247) variant was then verified. The plasmidcontaining the sequence encoding Gel_(c247) was designated pING3737(deposited on Jun. 9, 1992 with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md 20852 as ATCC Accession No. 69009).

(2-3) In the same manner, a cysteine residue was introduced in place ofthe amino acid at position 248 (a lysine) of gelonin with the mutagenicoligonucleotides GeloC-1 and GeloC-2 to generate analog Gel_(c248) inplasmid pING3741, and a cysteine residue was introduced at amino acidposition 239 (a lysine) with primers GeloC-9 and GeloC-10 to generateanalog Gel₂₃₉ in plasmid pING3744.

(4) Also in the same manner, a cysteine residue was introduced at aminoacid 244 (a lysine) of gelonin with mutagenic primers GeloC-5 andGeloC-6 to generate analog Gel_(c244) in the plasmid designatedpING3736. This variant was prepared by PCR using plasmid pING3734 astemplate DNA rather than pING3825. It therefore encodes the sameN-terminal gelonin amino acid sequence as plasmids pING3737, pING3741,and pING3744, but includes the PCR primer-derived 5'-end 32 nucleotidesinstead of the native gelonin 5'-end nucleotides.

(5) A cysteine residue was introduced in place of the amino acid (alysine) at position 10 of gelonin by a similar procedure. A cysteine wasintroduced with mutagenic primers GeloC-13 and GeloC-14 by amplifyingpING3824 with ara B2 (a vector primer) and GeloC-14, and in a separatereaction, with GeloC-13 and Gelo- 11. These reaction products were mixedand amplified with the outside primers ara B2 and Gelo-11. The PCRproduct was cut with PstI and NcoI, purified, and cloned back intopING3825 to generate analog Gel_(c10) in the plasmid designated pING3746(deposited on Jun. 9, 1992 with the American Type Culture Collection,12301 Parklawn Drive, Rockville, Md 20852 as ATCC Accession No. 69008).

(6) The asparagine at position 60 of gelonin was replaced with acysteine residue using two mutagenic oligos, GeloC-15 and GeloC-16, inconjunction with oligos ara B2 and Gelo-11 in the same manner as for theGel_(c10) variant. The plasmid encoding the Gel_(c60) analog wasdesignated pING3749.

(7) A cysteine was introduced at amino acid 103 (an isoleucine) by PCRwith mutagenic primers GeloC-20 and GeloC-21 in conjunction with primersara B2 and HINDIII-2. Template DNA (pING3733) was amplified withGeloC-21 and ara B2 and separately with GeloC-20 and HINDIII-2. Theproducts of these reactions were mixed and amplified with the outsideprimers ara B2 and HINDIII-2. The reaction product was cut with NcoI andBclI, purified, and inserted into pING3825 digested with NcoI and BclI.The oligonucleotides used to place a cysteine at residue 103 alsointroduced an AfllII restriction site which was verified in the clonedgene. The plasmid containing the Gel_(c103) analog was designatedpING3760.

(8) A cysteine was introduced at position 146 (an aspartic acid) by asimilar strategy. Template DNA (pING3733) was amplified with mutagenicprimer GeloC-22 and Gelo-14 and separately with mutagenic primerGeloC-23 and Gelo-19. The products of these reactions were mixed, andamplified with Gelo-19 and Gelo-14. The reaction product was cut withBgllI and EcoRI, and can be inserted into pING3825 in a three-pieceligation. The oligonucleotides used to place a cysteine at residue 146also introduced a NdeI restriction site which can be verified in thecloned gene.

(9) To introduce a cysteine at position 184 (an arginine) of gelonin,template DNA (pING3733) was amplified with mutagenic primer GeloC-25 andara B-2 and separately with mutagenic primer GeloC-24 and HINDIII-2. Theproducts of these reactions were mixed, and amplified with ara B2 andGelo-14. The reaction product was cut with NcoI and BclI, and insertedinto pING3825 previously digested with NcoI and BclI. Theoligonucleotides used to place a cysteine at residue 184 also introducedan NsiI restriction site which was verified in the cloned gene. Theplasmid containing the sequence encoding the Gel_(c184) variant wasdesignated pING3761.

(10) A cysteine can be introduced at position 215 (a serine) by asimilar strategy. Template DNA (pING3733) was amplified with mutagenicprimer GeloC-27 and ara B2 and separately with mutagenic primer GeloC-26and HINDIII-2. The products of these reactions were mixed, and amplifiedwith ara B2 and HINDIII2. The reaction product was cut with EcoRI andBclI, and can be inserted into pING3825 in a three-piece ligation.

(11 ) Another gelonin variant with a free cysteine residue was generatedby replacing one of the two naturally occurring gelonin cysteineresidues, the cysteine a position 50, with an alanine. Plasmid pING3824was amplified with primers GeloC-17 and Gelo-11, and concurrently in aseparate reaction with primers GeloC-19 and ara B2. The reactionproducts were mixed and amplified with ara B2 and Gelo-11. This productwas cut with NcoI and BglII, and cloned back into the vector portion ofpING3825 to generate pING3747 (ATCC 69101). This analog was designatedGel_(c44) because it contains a cysteine available for disulfide bondingat amino acid position 44.

(12) A gelonin variant with the natural cysteine at position 44 changedto alanine was constructed by amplifying pING3733 using the mutagenicoligos GeloC-28 and GeloC-29 in conjunction with primers ara B2 andHINDIII-2. The amplified DNA was cut with NcoI and BglII, and clonedinto a gelonin vector, generating pING3756. The variant generated wasdesignated Gel_(c50).

(13) A gelonin variant in which both the cysteine at position 44 and thecysteine at position 50 of gelonin were changed to alanine residues wasconstructed by overlap PCR of pING3824 using the mutagenic oligosGeloC-17 and GeloC-18 in conjunction with primers ara B2 and Gelo-11.This analog, like the native gelonin protein, has no cysteine residuesavailable for conjugation. The plasmid encoding the analog wasdesignated pING3750. The analog generated was designated Gel_(c44AC50A).

(14) The triple mutant GeloninC247_(c44AC50A) was constructed from theplasmids pING3824, pING3750 and pING3737. This variant contains anintroduced cysteine at position 247 while both of the naturallyoccurring cysteine residues at positions 44 and 50 have been replacedwith alanine and is desirable because disulfide linkage to an antibodyis assured at only a single cysteine residue in the gelonin analog.Plasmid pING3824 was cut with NcoI and XhoI and the vector fragment waspurified in an agarose gel. pING3750 was cut with NcoI and EcoRI andpING3737 was cut with EcoRI and XhoI. The NcoI-EcoRI fragment encodesthe alanines at positions 44 and 50 while the EcoRI-XhoI fragmentencodes the cysteine at position 247. Each of these fragments waspurified and ligated to the NcoI to XhoI vector fragment. The resultingplasmid is named pING3752.

(15) The triple mutant GeloninC10_(c44AC50A) was also constructed byassembly from previously assembled plasmids. In this case, pING3746 wascut with PstI and NcoI, while pING3750 was cut with NcoI and XhoI. Eachof the insert fragments were purified by electrophoresis in an agarosegel, and the fragments were ligated into a PstI and XhoI digested vectorfragment. The resulting vector was designated pING3753.

Each of the gelonin variants constructed was transformed into E. colistrain E 104. Upon induction of bacterial cultures with arabinose,gelonin polypeptide could be detected in the culture supernatants withgelonin-specific antibodies. There were no significant differencesdetected in the expression levels of gelonin from plasmids pING3734 andpING3825, or in the levels from any of the gelonin variants. Eachprotein was produced in E. coli at levels of approximately 1 g/l.

Example 4 Reticulocyte Lysate Assay

The ability of gelonin and recombinant gelonin analogs to inhibitprotein synthesis in vitro was tested using a reticulocyte lysate assay(RLA) described in Press et al., Immunol. Letters, 14, 37-41 (1986). Theassay measures the inhibition of protein synthesis in a cell-free systemusing endogenous globin mRNA from a rabbit red blood cell lysate.Decreased incorporation of tritiated leucine (³ HLeu) was measured as afunction of toxin concentration. Serial log dilutions of standard toxin(the 30 kD form of ricin A-chain, abbreviated as RTA 30), nativegelonin, recombinant gelonin (rGelonin) and gelonin analogs were testedover a range of 1 μg/ml to 1 pg/ml. Samples were tested in triplicate,prepared on ice, incubated for 30 minutes at 37° C., and then counted onan Inotec Trace 96 cascade ionization counter. By comparison with anuninhibited sample, the picomolar concentration of toxin (pM) whichcorresponds to 50% inhibition of protein synthesis (IC₅₀) wascalculated. As is shown in Table 2 below, recombinant gelonin and mostof its analogs exhibit activity in the RLA comparable to that of nativegelonin. For some of the analogs (such as Gel_(c239)), RLA activity wasdiminished.

                  TABLE 1                                                         ______________________________________                                        Toxin            IC.sub.50 (pM)                                               ______________________________________                                        RTA 30           2.5                                                          Gelonin          15                                                           rGelonin         11                                                           Gel.sub.C10      60                                                           Gel.sub.C44      20                                                           Gel.sub.C50      47                                                           Gel.sub.C60      26                                                           Gel.sub.C239     955                                                          Gel.sub.C244     32                                                           Gel.sub.C247     12                                                           Gel.sub.C248     47                                                           Gel.sub.C44AC50A 16                                                           GelC10.sub.C44AC50A                                                                            7                                                            GelC247.sub.C44AC50A                                                                           20                                                           ______________________________________                                    

Example 5 Preparation of Gelonin Immunoconjugates

Gelonin analogs of the invention were variously conjugated to murine(ATCC HB9286) and chimetic H65 antibody, (CH65) and cH65 antibodydomains (including cFab, cFab' and cF(ab')₂ fragments) that arespecifically reactive with the human T cell determinant CD5. H65antibody was prepared and purified by methods described in U.S. patentapplication Ser. No. 07/306,433, supra and International Publication No.WO 89/06968, supra. Chimetic H65 antibody was prepared according tomethods similar to those described in Robinson et at., Human Antibodiesand Hybridomas, 2, 84-93 (1991).

(1 ) Conjugation to H65 antibodies

To expose a reactive sulfhydryl, the unpaired cysteine residues of thegelonin analogs were first reduced by incubation with 0.1 to 2 mM DTT(30-60 minutes at room temperature), and then were desalted bysize-exclusion chromatography.

Specifically, the Gel_(c248) analog (3.8 mg/ml) was treated with 2 mMDTT for 60 minutes in 0.1 M NaPhosphate, 0.25 M NaC1, pH 7.5 buffer. TheGel_(c244) variant (7.6 mg/ml) was treated with 2 mM DTT for 30 minutesin 0.1 M NaPhosphate, 0.25 M NaC1, pH 7.5 buffer. The Gel_(c247) analog(4 mg/ml) was treated with 2 mM DTT for 30 minutes in 0.1 M NaPhosphate,0.5 M NaC1, pH 7.5 buffer with 0.5 mM EDTA. The Gel_(c239) variant (3.2mg/ml) was treated with 2 mM DTT for 30 minutes in 0.1 m NaPhosphate,0.5 M NaC1, pH 7.5 buffer with 0.5 mM EDTA. The Gel_(c44) analog (4.2mg/ml) was treated with 0.1 mM DTT for 30 minutes in 0.1 M NaPhosphate,0.1 M NaC1, pH 7.5 buffer with 0.5 mM EDTA. Lastly, the Gel_(c10)variant (3.1 mg/ml) was treated with 1 mM DTT for 20 minutes in 0.1 MNaPhosphate, 0.1 M NaC1, pH 7.5 buffer with 1 mM EDTA.

The presence of a free sulfhydryl was verified by reaction with DTNB andthe average value obtained was 1.4 ±0.65 SH/molecule. No free thiolswere detected in the absence of reduction.

H65 antibody and chimeric H65 antibody were chemically modified with thehindered linker 5-methyl-2-iminothiolane (M2IT) at lysine residues tointroduce a reactive sulfhydryl group as described in Goff et al.,Bioconjugate Chem., 1, 381-386 (1990).

Specifically, for conjugation with Gel_(c248) and Gel_(c244), murine H65antibody at 4 mg/mL was derivitized with 18x M2IT and 2.5 mM DTNB in 25mM TEOA, 150 mM NaC1, pH 8 buffer for 1 hour at 23° C. The reaction gave1.9 linkers per antibody as determined by DTNB assay.

For conjugation with Gel_(c247) and Gel_(c239), H65 antibody at 4.7mg/mL was derivitized with 20x M2IT and 2.5 mM DTNB in 25 mM TEOA 150 mMNaCl, pH 8 buffer for 50 minutes at 23° C. The reaction gave 1.6 linkersper antibody as determined by DTNB assay.

Before reaction with Gel_(c44), H65 antibody at 5.8 mg/mL wasderivitized with 20x m2IT and 2.5 mM DTNB in 25 mM TEOA, 150 mM NaC1, pH8 buffer for 30 minutes at 23°C. The reaction gave 1.5 linkers perantibody as determined by DTNB assay.

For conjugation with Gel_(c10), H65 antibody at 2.2 mg/mL wasderivitized with 10x m2IT and 2.5 mM DTNB in 25 mM TEOA, 150 mM NaC1, pH8 buffer for 1 hour at 23° C. The reaction gave 1.4 linkers per antibodyas determined by DTNB assay.

Chimeric H65 antibody was prepared for conjugation in a similar mannerto murine H65 antibody.

Two methods were initially compared for their effectiveness in preparingimmunoconjugates with recombinant gelonin. First, the native disulfidebond in recombinant gelonin was reduced by the addition of 2mM DTT atroom temperature for 30 minutes. The reduced gelonin was recovered bysize-exclusion chromatography on a column of Sephadex GF-05LS andassayed for the presence of free sulfhydryls by the DTNB assay. 1.4 freeSH groups were detected. This reduced gelonin was then reacted withH65-(M2IT)-S-S-TNB (1.8 TNB groups/H65). Under these experimentalconditions, little or no conjugate was prepared between reduced geloninand thiol-activated H65 antibody.

In contrast, when both the recombinant gelonin and the H65 antibody werefirst derivitized with the crosslinker M2IT (creating gelonin-(M2IT)-SHand H65-(M2IT)-S-S-TNB) and then mixed together,H65-(M2IT)-S-S-(M2IT)-gelonin conjugate was prepared in good yield(toxin/antibody ratio of 1.6). The starting materials for thisconjugation (gelonin-(M2IT)-SH and H65-(M2IT)-S-S-TNB) containedlinker/protein ratios of 1.2 and 1.4, respectively. Native gelonin wasderivatized in a similar manner prior to conjugation to murine orchimeric H65 antibody.

The reduced gelonin analogs were mixed with H65-(M2IT)-S-S-TNB to allowconjugation. The following conjugation reactions were set up for eachanalog: 23 mg (in 7.2 ml) of H65-M2IT-TNB were mixed with a 5-fold molarexcess of Gel_(C248) (23 mg in 6 ml) for 2 hours at room temperature,then for 18 hours overnight at 4° C.; 23 mg (in 7.3 ml) of H65-m2IT-TNBwere mixed with a 5-fold molar excess of Gel_(C244) (23 mg in 3 ml) for3 hours at room temperature, then for 18 hours overnight at 4° C.; 9 mg(in 2.8 mL) of H65-m2IT-TNB were mixed with a 5-fold molar excess ofGel_(C247) (9 mg in 2.25 mL) for 2 hours at room temperature, then for 5nights at 4° C.; 9 mg (in 2.8 mL) of H65-m2IT-TNB were mixed with a5-fold molar excess of Gel_(C239) (9mg in 2.6 mL) for 2 hours at roomtemperature, then at 4° C. for 3 days; 12 mg (in 1.9 mL) of H65-m2IT-TNBwere mixed with a 5.6-fold molar excess of Gel_(C44) (13.44 mg in 3.2mL) for 4.5 hours at room temperature, then 4° C. overnight; and 11 mgof H65-m2IT-TNB were mixed with a 5-fold molar excess of Gel_(C10) (11mg in 3.5 mL) for 4 hours at room temperature, then at 4° C. overnight.

Following conjugation, unreacted M2IT linkers on the antibody werequenched with 1:1 mole cysteamine to linker for 15 minutes at roomtemperature. The quenched reaction solution was then loaded onto a gelfiltration column [Sephadex G-150 (Pharmacia) in the case of Gel_(C248),GelC₂₄₇, Gel_(C244) and Gel_(C239) and an AcA-44 column (IBF Biotecnics,France) in the case of Gel_(C44) and Gel_(C10) ]. The reactions were runover the gel filtration columns and eluted with 10 mM Tris, 0.15M NaClpH 7. The first peak off each column was loaded onto Blue Toyopearl®resin (TosoHaas, Philadelphia, Pa.) in 10 mM Tris, 30 mM NaCl, pH 7 andthe product was eluted with 10 mM Tris, 0.5M NaCl, pH 7.5.

Samples of the final conjugation products were run on 5% non-reduced SDSPAGE, Coomassie stained and scanned with a Shimadzu laser densitometerto quantitate the number of toxins per antibody (T/A ratio). The yieldof final product for each analog conjugate was as follows: Gel_(C248),17 mg with a T/A ration of 1.6; Gel_(C247), 1.1 mg with a T/A ratio of1; Gel_(C244), 4.5 mgs with a T/A ratio of 1.46; Gel_(C239), 2.9 mg witha T/A ratio of 2.4; Gel_(C44), 7.3 mg with a T/A ratio of 1.22; andGel_(C10), 6.2 mg with a T/A ratio of 1.37. Conjugation efficiency(i.e., conversion of free antibody to immunoconjugate) was significantlygreater (˜80%) for some analogs (Gel_(C10), Gel_(C44), Gel_(C239),Gel_(C247), and Gel_(C248)) than for others (˜10%, Gel_(C244)).

(2) Conjugation to antibody fragments

Analogs Gel_(C247) and Gel_(C44) were conjugated to various chimeric[cFab, cFab' and cF(ab')₂ ] and "human engineered"[he1 Fab, he2 Fab, he3Fab, he1 Fab' and he1 F(ab')₂ ] antibody fragments. Chimetic H65antibody fragments may be prepared according to the methods described inU.S. patent application Ser. No. 07/714,175, supra and in InternationalPublication No. WO 89/00999, supra. The DNA sequences encoding thevariable regions of H65 antibody fragments that were human engineered(referring to the replacement of selected murine-encoded amino acids tomake the H65 antibody sequences less immunogenic to humans) according tothe methods described in co-pending, co-owned U.S. Pat. application Ser.No. 07/808,454 filed Dec. 13, 1991 which is incorporated by referenceherein, are set out in SEQ ID NO: 71 (the kappa chain of he1 and he2),SEQ ID NO: 72 (the gamma chain of he1), SEQ ID NO: 73 (the gamma chainof he2 and he3) and SEQ ID NO: 74 (the kappa chain of he3)

The chimetic H65 antibody fragments were conjugated to Gel_(C247) analogbasically as described below for conjugation of human engineered Fab andFab' fragments to Gel_(C247) and Gel_(C44).

(a) he1 Fab-Gel_(C247)

The he1 Fab was dialyzed into 25 mM TEOA buffer, 250 mM NaCl, pH 8 andthen concentrated to 6.8 mg/mL prior to derivitization with the M2ITcrosslinker. For the linker reaction, M2IT was used at 20-fold molarexcess, in the presence of 2.5 mM DTNB. The reaction was allowed toproceed for 30 minutes at room temperature, then desalted on GF05 (gelfiltration resin) and equilibrated in 0.1 M Na Phosphate, 0.2M NaCl, pH7.5. A linker number of 1.8 linkers per Fab was calculated based on theDTNB assay. The he1 Fab-M2IT-TNB was concentrated to 3.7 mg/mL prior toconjugation with Gel_(C247).

Gel_(C247) at 12.8 mg/mL in 10 mM Na Phosphate, 0.3M NaCl, was treatedwith 1 mM DTT, 0.5 mM EDTA for 20 minutes at room temperature to exposea reactive sulfhydryl for conjugation and then was desalted on GF05 andequilibrated in 0.1M Na Phosphate, 0.2M NaCl, pH 7.5. Free thiol contentwas determined to be 0.74 moles of :free SH per mole of Gel_(C247) usingthe DTNB assay. The gelonin was concentrated to 8.3 mg/mL prior toconjugation with activated antibody.

The conjugation reaction between the free thiol on Gel_(C247) and thederivitized hel Fab-M2IT-TNB, conditions were as follows. A 5-foldexcess of the gelonin analog was added to activated he 1 Fab-M2IT-TNB(both proteins were in 0.1M Na Phosphate, 0.2M NaCl, pH7.5) and thereaction mixture was incubated for 3.5 hours at room temperature andthen overnight at 4° C. Following conjugation, untreated M2IT linkerswere quenched with 1:1 mole cysteamine to linker for 15 minutes at roomtemperature. The quenched reaction solution was loaded onto a gelfiltration column (G-75) equilibrated with 10 mM Tris, 150 mM NaCl, pH7. The first peak off this column was diluted to 30 mM NaCl with 10 mMTris, pH7 and loaded on Blue Toyopearl®. The product was eluted with 10mM Tris, 0.5M NaCl, pH 7.5.

(b) he1 Fab '-Gel_(C247)

Similarly, the H65 he1 Fab' fragment was dialyzed into 25 mM TEOAbuffer, 400 mM NaCl, pH 8 at 2.9 mg/mL prior to derivitization with theM2IT crosslinker. For the linker reaction, M2IT was used at 20-foldmolar excess, in the presence of 2.5 mM DTNB. The reaction was allowedto proceed for 1 hour at room temperature then it was desalted on GF05(gel filtration resin) and equilibrated in 0.1 M Na Phosphate, 0.2MNaCl, pH 7.5. A linker number of 1.6 linkers per Fab' was calculatedbased on the DTNB assay. The he1 Fab'-M2IT-TNB was concentrated to 3.7mg/mL prior to conjugation with Gel_(C247)

The Gel_(C247) at 77 mg/mL was diluted with in 10 mM Na Phosphate, 0.1MNaCl to a concentration of 5 mg/mL, treated with 1 mM DTT, 0.5 mM EDTAfor 30 minutes at room temperature to expose a free thiol forconjugation and then was desalted on GF05 and equilibrated in 0.1M NaPhosphate, 0.2M NaCl, pH 7.5. Free thiol content was determined to be1.48 moles of free SH per mole of Gel_(C247) using the DTNB assay. TheGel_(C247) was concentrated to 10 mg/mL prior to conjugation withactivated he1 Fab'-M2IT-TNB.

For the reaction between the free thiol on Gel_(C247) and thederivitized he1 Fab'-M2IT-TNB, conditions were as follows. A 5.7-foldmolar excess of gelonin was added to activated he1 Fab'-M2IT-TNB and thefinal salt concentration was adjusted to 0.25M. The reaction mix wasincubated for 1.5 hours at room temperature and then over the weekend at4° C. Following conjugation, unreacted M2IT linkers were quenched with1:1 mole cysteamine to linker for 15 minutes at room temperature. Thequenched reaction solution was loaded onto a gel filtration column(AcA54) equilibrated with 10 mM Tris, 250 mM NaCI, pH 7.5. The firstpeak off this column was diluted to 20 mM NaCl with 10 mM Tris, pH 7 andloaded on Blue Toyopearl® which was equilibrated in 10 mM Tris, 20 mMNaCl, pH 7. The column was then washed with 10 mM Tris, 30 mM Nacl, pH7.5. The product was eluted with 10 mM Tris, 1M NaCl, pH 7.5.

(c) he2 Fab Gel_(C44)

The he2 Fab was dialyzed overnight into 25 mM TEOA, 0.25M NaCl, pH 8buffer and then concentrated to 13.3 mg/mL prior to derivitization withthe M2IT crosslinker. For the linker reaction, M2IT was used in a20-fold molar excess in the presence of 2.5 mM DTNB. The reaction wasallowed to proceed for 20 minutes at room temperature and was thenalesalted on a GF05-LS (gel filtration) column, equilibrated in 0.1M NaPhosphate, 0.2M NaCl with 0.02% Na azide. A linker number of 1.7 linkersper Fab-M2IT-TNB was calculated based on the DTNB assay. Afterderivitization and gel filtration, the he2 Fab concentration was 5.2mg/mL.

Gel_(C44) at 8.33 mg/mL in 10 mM Na Phosphate, pH 7.2 was treated with 5mM DTT and 0.5 mM EDTA for 30 minutes at room temperature to expose areactive thiol for conjugation and then was desalted on GF05-LS resinequilibrated in 0.1M Na Phosphate, 0.1M NaCl with 0.5 mM EDTA plus 0.02% Na azide, pH 7.5. Free thiol content was determined to be 0.83 molesof free SH per mole of Gel_(C44) using the DTNB assay. The gelonin wasconcentrated to 11.4 mg/mL prior to conjugation with activated he2 Fab.

The conjugation reaction conditions between the free thiol on Gel_(C44)and the derivitized he2 Fab-M2IT-TNB were as follows. A 3-fold excess ofthe gelonin analog was added to activated he2 Fab-M2IT-TNB (bothproteins were in 0.1 M Na Phosphate, 0.1M NaCl, pH 7.5 but the geloninsolution contained 0.5 mM EDTA as well). The reaction mixture wasconcentrated to half its original volume, then the mixture was incubatedfor 4 hours at room temperature followed by 72 hours at 4° C. Followingthe incubation period the efficiency of conjugation was estimated at70-75 % by examination of SDS PAGE.

Following conjugation the excess M2IT linkers were quenched byincubation with 1:1 mole cysteamine to linker for 15 minutes at roomtemperature. The quenched reaction as loaded onto a gel filtrationcolumn (G-75) equilibrated in 10 mM Tris, 0.15M NaCl, pH 7. The firstpeak off this column was diluted to 30 mM NaCl with 10 mM Tris, pH 7 andloaded onto a Blue Toyopearl® (TosoHaas) column. The product was elutedwith 10 mM Tris, 1M NaCl, pH 7.5.

(d) he3 Fab Gel_(C44)

Similarly, the he3 Fab was dialyzed overnight into 25 mM TEOA, 0.25MNaCl, pH 8 buffer and then concentrated to 5 mg/mL prior toderivitization with the M2IT crosslinker. For the linker reaction, M2ITwas used in a 10-fold molar excess in the presence of 2.5 mM DTNB. Thereaction was allowed to proceed for 45 minutes at room temperature andwas then alesalted on a GF05-LS (gel filtration) column, equilibrated in0.1M Na Phosphate, 0.2M NaCl with 0.02% Na azide. A linker number of1M2IT per Fab-M2IT-TNB was calculated based on the DTNB assay. Afterderivitization and gel filtration, the he3 Fab concentration was 5.3mg/mL.

Gel_(C44) at 7.8 mg/mL in 0.1M Na Phosphate, 0.1M NaCl, pH 7.5 wastreated with 1.5 mM DTT and 1 mM EDTA for 30 minutes at room temperatureto expose a reactive thiol for conjugation and then was desalted onGF05-LS resin equilibrated in 0.1M Na Phosphate, 0.1M NaCl plus 0.02% Naazide, pH 7.5. Free thiol content was determined to be 0.66 moles offree SH per mole of Gel_(C44) using the DTNB assay. The gelonin wasconcentrated to 5.2 mg/mL prior to conjugation with activated he3 Fab.

The conjugation reaction conditions between. the free thiol on Gel_(C44)and the derivitized he3 Fab-M2IT-TNB were as follows. A 5-fold excess ofthe gelonin analog was added to activated he3 Fab-M2IT-TNB (bothproteins were in 0.1M Na phosphate 0.1M NaCl, pH 7.5). The reactionmixture was incubated for 2 hours at room temperature followed by 72hour at 4° C. Following the incubated period the efficiency ofconjugation was estimated at 70-75 % by examination of SDS PAGE.

Following conjugation, the excess M2IT linkers were quenched byincubation with 1:1 mole cysteamine to linker for 15 minutes at roomtemperature. The quenched reaction was loaded onto a GammaBind G(immobilized protein G affinity resin, obtained from Genex,Gaithersburg, Md.) equilibrated in 10 mM Na Phosphate, 0.15M NaCl, pH 7.It was eluted with 0.5M NaOAc, pH 3 and neutralized with Tris. It wasdialyzed into 10 mM Tris, 0.15M NaCl, pH 7 overnight, then diluted to 30mM NaCl with 10 mM Tris, pH 7 and loaded onto a blue Toyopearl®(TosoHaas) column. The product was eluted with 10 mM Tris, 1M NaCl, pH7.5

EXAMPLE 6 Whole Cell Kill Assays

Immunoconjugates prepared with gelonin and gelonin analogs were testedfor cytotoxicity against an acute lymphoblastoid leukemia T cell line(HSB2 cells) and against human peripheral blood mononuclear cells(PBMCs). Immunoconjugates of ricin A-chain with H65 antibody (H65-RTA)and antibody fragments were also tested. The ricin A-chain (RTA) as wellas the H65-RTA immunoconjugates were prepared and purified according tomethods described in U.S. patent application Ser. No. 07/306,433, supraand in International Publication No. WO 89/06968, Supra.

Briefly, HSB2 cells were incubated with immunotoxin and the inhibitionof protein synthesis in the presence of immunotoxin was measuredrelative to untreated control cells. The standard immunoconjugatesH65-RTA (H65 derivitized with SPDP linked to RTA), H65-Gelonin andH65-rGelonin, H65 fragment immunoconjugate, and gelonin immunoconjugatesamples were diluted with RPMI without leucine at half-logconcentrations ranging from 2000 to 0.632 ng/ml. All dilutions wereadded in triplicate to microtiter plates containing 1×10⁵ HSB2 cells.HSB2 plates were incubated for 20 hours at 37° C. and then pulsed with ³H-Leu for 4 hours before harvesting. Samples were counted on the InotecTrace 96 cascade ionization counter. By comparison with an untreatedsample, the picomolar concentration (pM) of immunotoxin which resultedin a 50% inhibition of protein synthesis (IC₅₀) was calculated. In orderto normalize for conjugates containing differing amounts of toxin ortoxin analog, the cytotoxicity data were converted to picomolar toxin(pM T) by multiplying the conjugate IC₅₀ (in pM) by the toxin/antibodyratio which is unique to each conjugate preparation.

The PMBC assays were performed as described by Fishwild et al., Clin.and Exp. Immunol., 86, 506-513 (1991) and involved the incubation ofimmunoconjugates with PBMCs for a total of 90 hours. During the final 16hours of incubation, ³ H-thymidine was added; upon completion,immunoconjugate-induced inhibition of DNA synthesis was quantified. Theactivities of the H65 and chimetic H65 antibody conjugates against HSB2cells and PBMC cells are listed in Table 2 below.

                  TABLE 2                                                         ______________________________________                                        IC.sub.50 (PM T)                                                              Conjugate            HSB2 Cells                                                                              PBMCs                                          ______________________________________                                        H65-RTA              143       459                                            H65-(M2IT)-S--S-(M2IT)-Gelonin                                                                     1770      81                                             H65-(M2IT)-S--S-(M2IT)-rGelonin                                                                    276       75                                             H65-(M2IT)-S--S-Gel.sub.C10                                                                        140       28                                             H65-(M2IT)-S--S-Gel.sub.C44                                                                        99        51                                             H65-(M2IT)-S--S-Gel.sub.C239                                                                       2328      180                                            H65-(M2IT)-S--S-Gel.sub.C244                                                                       >5000     >2700                                          H65-(M2IT)-S--S-Gel.sub.C247                                                                       41        35                                             H65-(M2IT)-S--S-Gel.sub.C248                                                                       440       203                                            cH65-RTA.sub.30      60        400                                            cH65-(M21T)-S--S-(M2IT)-Gelonin                                                                    1770      140                                            cH65-(M2IT)-S--S-(M2IT)-rGelonin                                                                   153       120                                            cH65-(M2IT-S--S-Gel.sub.C239                                                                       >7000     290                                            cH65-(M2IT-S--S-Gel.sub.C247                                                                       34        60                                             cH65-(M21T)-S--S-Gel.sub.C248                                                                      238       860                                            ______________________________________                                    

Against HSB2 cells, many of the gelonin analog immunoconjugates weresignificantly more potent than conjugates prepared with native geloninor recombinant, unmodified gelonin, both in terms of a low IC₅₀ value,but also in terms of a greater extent of cell kill. Against human PBMCs,the gelonin analog conjugates were at least as active as native andrecombinant gelonin conjugates. Importantly, however, some of theconjugates (for example, Gel_(C10), Gel_(C44) and Gel_(C247)) exhibitedan enhanced potency against PBMCs compared to native and recombinantgelonin conjugates, and also exhibited an enhanced level of cell kill(data not shown).

The activities of the H65 antibody fragment conjugates against HSB2cells and PBMC cells are listed in Tables 3 and 4 below, wherein extentof kill in Table 4 refers to the percentage of protein synthesisinhibited in HSB2 cells at the highest immunotoxin concentration tested(1μg/ml).

                  TABLE 3                                                         ______________________________________                                        IC.sub.50 (pM T)                                                              Conjugate       HSB2 Cells                                                                              PBMCs                                               ______________________________________                                        cFab'-RTA 30    530       1800                                                cFab'-rGelonin  135       160                                                 cFab'-Gel.sub.C247                                                                             48       64                                                  cF(ab').sub.2 -RTA 30                                                                          33       57                                                  cF(ab').sub.2 -rGelonin                                                                        55       34                                                  cF(ab').sub.2 Gel.sub.C247                                                                     23       20                                                  cF(ab').sub.2 -Gel.sub.C248                                                                   181       95                                                  ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        IC.sub.50 (pM T)                                                              Conjugate       HSB2 Cells                                                                              Extent of Kill                                      ______________________________________                                        hel Fab'-Gel.sub.C247                                                                         57.7      93%                                                 hel Fab-Gel.sub.C247                                                                          180       94%                                                 he2 Fab-Gel.sub.C44                                                                           363       91%                                                 he3 Fab-Gel.sub.C44                                                                           191       93%                                                 cFab'-Gel.sub.C247                                                                            47.5      93%                                                 cF(ab').sub.2 -rGelonin                                                                       45.4      85%                                                 F(ab').sub.2 -Gel.sub.C247                                                                    77.5      83%                                                 cF(ab').sub.2 -Gel.sub.C247                                                                   23.2      85%                                                 ______________________________________                                    

The cFab '-Gel₂₄₇ immunoconjugate is clearly more cytotoxic than cFab'conjugates with recombinant gelonin or RTA 30.

EXAMPLE 7 Solubility

Recombinant gelonin and the gelonin analogs exhibited enhancedsolubility in comparison to both native gelonin and RTA30. In addition,recombinant gelonin and gelonin analog immunoconjugates exhibitedenhanced solubility relative to immunoconjugates prepared with nativegelonin and RTA30. This enhanced solubility was particularly noteworthyfor recombinant gelonin and analog conjugates prepared with chimeric Fabfragments.

Disulfide Bond Stability Assay

The stability of the disulfide bond linking a RIP to a targetingmolecule (such as an antibody) is known to influence the lifespan ofimmunoconjugates in vivo [see Thorpe et al., Cancer Res., 47, 5924-5931(1987)]. For example, conjugates in which the disulfide bond is easilybroken by reduction in vitro are less stable and less efficacious inanimal models [see Thorpe et al., Cancer Res., 48, 6396-6403 (1988)].

Immunoconjugates prepared with native gelonin, recombinant gelonin andgelonin analogs were therefore examined in an in vitro disulfide bondstability assay similar to that described in Wawrzynczak et al., CancerRes., 50, 7519-7526 (1990). Conjugates were incubated with increasingconcentrations of glutathione for 1 hour at 37° C. and, afterterminating the reaction with iodoacetamide, the amount of RIP releasedwas quantitated by size-exclusion HPLC on a TosoHaas TSK-G2000SW column.

By comparison with the amount of RIP released by high concentrations of2-mercaptoethanol (to determine 100% release), the concentration ofglutathione required to release 50% of the RIP (the RC₅₀) wascalculated. The results of assays for H65 antibody conjugates are setout in Table 5 below.

                  TABLE 5                                                         ______________________________________                                        Conjugate             RC.sub.50 (mM)                                          ______________________________________                                        H65-RTA 30            3.2                                                     H65-(M2IT)-S--S-(M2IT)-gelonin                                                                      11.1                                                    H65-(M2IT)-S--S-(M2IT)-rGelonin                                                                     3.0                                                     H65-(M2IT)-S--S-Gel.sub.C10                                                                         2.5                                                     H65-(M2IT)-S--S-Gel.sub.C44                                                                         0.6                                                     H65-(M2IT)-S--S-Gel.sub.C239                                                                        774.0                                                   H65-(M2IT)-S--S-Gel.sub.C244                                                                        1.2                                                     H65-(M2IT)-S--S-Gel.sub.C247                                                                        0. 1                                                    H65-(M2IT)-S--S-Gel.sub.C248                                                                        0.4                                                     cH65-RTA 30           2.50                                                    cH65-(M2IT)-S--S-(M2IT)-rGelonin                                                                    2.39                                                    cH65-(M2IT)-S--S-Gel.sub.C247                                                                       0.11                                                    cH65-(M2IT)-S--S-Gel.sub.C248                                                                       0.32                                                    ______________________________________                                    

The foregoing results indicate that the stability of the bonds betweenthe different gelonin proteins and H65 antibody varied greatly. With theexception of Gel_(C10) and Gel_(C239), most of the gelonin analogsresulted in conjugates with linkages that were somewhat less stable inthis in vitro assay than the dual-linker chemical conjugate. Thestability of the Gel_(C239) analog, however, was particularly enhanced.

The results of the assay for H65 antibody fragment conjugates are setout in Table 6 below.

                  TABLE 6                                                         ______________________________________                                        Conjugate        RC.sub.50 (mM)                                               ______________________________________                                        he1 Fab'-Gel.sub.C247                                                                          0.07                                                         cFab'-Gelonin    1.27                                                         cFab'-Gel.sub.C247                                                                             0.08                                                         cF(ab').sub.2 -RTA 30                                                                          1.74                                                         cF(ab').sub.2 -rGelonin                                                                        2.30                                                         cF(ab').sub.2 -Gel.sub.C247                                                                    0.09                                                         cF(ab').sub.2 -Gel.sub.C248                                                                    0.32                                                         he2 Fab-Gel.sub.C44                                                                            0.46                                                         he3 Fab-Gel.sub.C44                                                                            0.58                                                         ______________________________________                                    

From the RC₅₀ results presented in Tables 5 and 6, it appears that theparticular RIP analog component of each immunotoxin dictates thestability of the immunotoxin disulfide bond in vitro.

EXAMPLE 8

Pharmacokinetics of Conjugates to H65 Antibody

The pharmacokinetics of gelonin analogs Gel_(C247), Gel_(C44) andGel_(C10) linked to whole H65 antibody was investigated in rats. An IVbolus of 0.1 mg/kg of ¹²⁵ I-labelled immunoconjugateH65-(M2IT)-S-S-Gel_(C247), H65-(M2IT)-S-S-Gel_(C44) orH65-(M2IT)-S-S-Gel_(C10) was administered to male Sprague-Dawley ratsweighing 134-148 grams. Serum samples were collected from the rats at 3,15, 30 and 45 minutes, and at 1.5, 2, 4, 6, 8, 18, 24, 48, 72, and 96hours. Radioactivity (cpm/ml) of each sample was measured, and SDS-PAGEwas performed to determine the fraction of radioactivity associated withwhole immunoconjugate. Immunoconjugate associated serum radioactivitywas analyzed using the computer program PCNONLIN (SCI Software,Lexington, Ky.). Table 7 below lists the pharmacokinetic parameters ofthe immunoconjugates. In the table, the standard error for each value isindicated and a one way analysis of variance is presented, IC is theimmunoconjugate (specified by the abbreviation for the gelonin variantthat is part of the immunoconjugate) n is the number of animals in thestudy, Vc is the central volume of distribution, Cl is the clearance,MRT is the total body-mean residence time, Alpha is the α half-life andBeta is the β half-life of the immunoconjugate.

                  TABLE 7                                                         ______________________________________                                                Vc       Cl        MRT    Alpha Beta                                  IC      (ml/kg)  (ml/hr/kg)                                                                              (hours)                                                                              (hours)                                                                             (hours)                               ______________________________________                                        H65     65.3 ±                                                                              11.0 ± 16.5 ±                                                                            2.3 ±                                                                            20.5 ±                             Gel.sub.C247                                                                          3.4      0.4       1.9    0.2   3.0                                   n = 32                                                                        H65 Gel.sub.C44                                                                       61.9 ±                                                                               4.1 ± 22.7 ±                                                                            3.0 ±                                                                            17.8 ±                             n = 38  2.4      0.1       0.7    0.7   0.8                                   H65 Gel.sub.C10                                                                       59.2 ±                                                                               2.5 ± 42.7 ±                                                                            3.3 ±                                                                            32.9 ±                             n = 45  1.3      0.04      1.1    0.3   1.1                                   p-value 0.176    <0.0001   <0.0001                                                                              0.303 <0.0001                               ______________________________________                                    

The Gel_(C247) immunoconjugate was found to have α and β half lives of2.3 and 20 hours, with a total mean residence time of 17 hours. The 72and 96 hour time points were excluded from analysis because of the poorresolution of immunoconjugate associated radioactivity on the SDS-PAGEgel for these serum samples.

Because in vitro studies suggested that the Gel_(C10) immunoconjugatehad greater disulfide bond stability, it was anticipated that its halflives in vivo would be longer relative to the cys₂₄₇ form of theimmunoconjugate. The β half life of the immunoconjugate was about 33hours compared to 20 hours for the Gel_(C247) conjugate. The total meanresidence time was also much greater for the Gel_(C10) immunoconjugate(42 hours versus 42 hours for the Gel₂₄₇ conjugate). In addition, theclearance of the Gel_(C10) immunoconjugate was 2.5 ml/hr/kg, about fourtimes less than that of the Gel_(C247) immunoconjugate (11 ml/hr/kg). Asalso predicted from the in vitro disulfide stability data, the clearanceof the Gel_(C44) immunoconjugate was intermediate between those of theGel_(C10) and Gel_(C247) immunoconjugates.

Based on these studies, the Gel_(C10) analog conjugated to H65 antibodyhas greater in vivo stability than the Gel_(C44) and Gel_(C247) analogsconjugated to H65 antibody (as determined by the longer mean residencetime and clearance rates), although the properties of the Gel_(C44)immunoconjugate more closely resembled those of the Gel_(C10)immunoconjugate than the Gel_(C247) immunoconjugate.

Pharmacokinetics of Conjugates to H65 Antibody Fragments

The pharmacokinetics of Gel_(C247) and Gel_(C44) analogs linked to humanengineered H65 Fab fragments were also investigated in rats. An IV bolusof 0.1 mg/kg of ¹²⁵ l-labelled he1 H65 Fab-Gel_(C247), he2 H65Fab-Gelc44 or he3 H65 Fab-Gel_(C44) was administered to maleSprague-Dawley rats weighing 150-180 grams. Serum samples were collectedat 3, 5, 15, 20, 30, and 40 minutes, and 1, 1.5, 3, 6, 8, 18, 24, 32,48, and 72 hours, and were analyzed by ELISA using rabbit anti-Geloninantibody as the capture antibody and biotin-labelled goat anti-humankappa light chain antibody as the secondary antibody. Results of theanalysis are presented in Table 8 below. In the table, the standarderror for each value is shown, and IC is the immunoconjugate, n is thenumber of animals in the study, Vc is the central volume ofdistribution, Vss is the steady state volume of distribution, Cl is theclearance, MRT is the total body mean residence time, Alpha is the αhalf-life and Beta is the β half-life of the indicated conjugate.

                                      TABLE 8                                     __________________________________________________________________________           Vc   Vss   Cl    MRT  Alpha Beta                                       IC     (ml/kg)                                                                            (ml/hr/kg)                                                                          (ml/hr/kg)                                                                          (hours)                                                                            (hours)                                                                             (hours)                                    __________________________________________________________________________    he1 Gel.sub.C247                                                                     48 ± 3                                                                          133 ± 7                                                                          62 ± 3                                                                           2.1 ± 0.1                                                                       0.33 ± 0.03                                                                      3.0 fixed                                  n = 27                                                                        he2 Gel.sub.C44                                                                      54 ± 5                                                                          141 ± 8                                                                          53 ± 3                                                                           2.7 ± 0.2                                                                       0.37 ± 0.04                                                                      3.1 fixed                                  n = 28                                                                        he3 Gel.sub.C44                                                                      77 ± 6                                                                          140 ± 20                                                                         57 ± 3                                                                           2.5 ± 0.4                                                                       0.58 ± 0.11                                                                      3.0 ± 1.0                               n = 33                                                                        __________________________________________________________________________

Comparing the three immunoconjugates, the pharmacokinetics of he1 H65Fab-Gel_(C247), he2 H65 Fab-Gel_(C44) and he3 Fab-Gel_(C44) were verysimilar, having similar alpha and beta half-lives, mean residence times,and clearance, particularly when comparing parameters obtained from theELISA assayed curves. This is in contrast to their whole antibodyimmunoconjugate counterparts, where the clearance of Gel_(C247)immunoconjugate (11 ml/kg/hr) was three-fold greater than that ofGel_(C44) immunoconjugate (4 ml/kg/hr). This suggests that cleavage ofthe disulfide bond linking the Fab fragment and gelonin is not asimportant for the serum clearance of Fab immunoconjugates as for wholeantibody immunoconjugates.

Immunogencity of Immunoconjugates

Outbred Swiss/Webster mice were injected repeatedly (0.2 mg/kg eachinjection) with murine H65 antibody conjugates prepared with RTA, RTA30and recombinant gelonin. The cycle was such that each animal wasinjected on days 1 and 2, and then the injections were repeated 28 and29 days later. The animals received 5 such cycles of injections. Oneweek and three weeks following each series of injections, blood wascollected and the amount of anti-RIP antibodies present was determinedby ELISA; peak titers for each cycle are shown in Table 9. RTA and RTA30generated strong responses which began immediately following the firstcycle of injections and remained high throughout the experiment. Incontrast, no immune response was detected for the gelonin conjugate,even after 5 cycles of injections. When the conjugates were mixed withComplete Freund Adjuvant and injected i.p. into mice, anti-RTA andRTA-30 antibodies were readily detected after several weeks. These dataindicate that anti-gelonin antibodies, if generated, would have beendetected by the ELISA assay, and suggest that recp,bomamt gelonin may bemuch less immunogenic in animals than is RTA.

                  TABLE 9                                                         ______________________________________                                        Cycle    H65-RTA      H65-RTA30  H65-rGel                                     ______________________________________                                        Prebleed  100          100       100                                          Cycle 1   168          117       100                                          Cycle 2  4208         1008       100                                          Cycle 3  7468         3586       100                                          Cycle 4  5707         3936       100                                          Cycle 5  4042         2505       100                                          ______________________________________                                    

EXAMPLE 9

A human peripheral blood lymphocyte (PBL)-reconstituted, severe combinedimmunodeficient mouse model was utilized to evaluate the in vivoefficacy of various immunoconjugates comprising the gelonin analogsGel_(C247) and Gel_(C44). Immunoconjugates were tested for the capacityto deplete human blood cells expressing the CD5 antigen.

Human PBL Donors and Cell Isolation

Human peripheral blood cells were obtained from lymphapheresis samples(HemaCare Corporation, Sherman Oaks, Calif.) or venous blood samples(Stanford University Blood Bank, Palo Alto, Calif.) collected fromhealthy donors. Blood cells were enriched for PBLs using Ficoll-Hypaquedensity gradient centrifugation (Ficoll-Paque®; Pharmacia, Piscataway,N.J.) and subsequently washed 4 times with PBS. Residual erythrocyteswere lysed with RBC lysing buffer (16 μM ammonium chloride, 1 mMpotassium bicarbonate, 12.5 μM EDTA) during the second wash. Cellviability in the final suspension was >95% as assessed by trypan bluedye exclusion.

Animals and Human PBL Transfer

CB.17 scid/scid (SCID) mice were purchased from Taconic (Germantown,N.Y.) or were bred under sterile conditions in a specific pathogen-freeanimal facility (original breeding pairs were obtained from HanaBiologics, Alameda, Calif.). Animals were housed in filter-top cages andwere not administered prophylactic antibiotic treatment. Cages, bedding,food and water were autoclaved before use. All manipulations withanimals were performed in a laminar flow hood.

Untreated SCID mice were bled for determination of mouse Ig levels.Human PBL-injected mice were bled at various intervals for quantitationof human Ig and slL-2R. Blood collection was from the retro-orbitalsinus into heparinized tubes. Blood samples were centrifuged at 300×gfor 10 min, and plasma was collected and stored at -70° C. Mouse andhuman Ig were quantified using standard sandwich ELISAs. Briefly,flat-bottom microtiter plates (MaxiSorp Immuno-Plates, Nunc, Roskilde,Denmark) were coated overnight at 4° C. with goat anti-mouse IgG+IgA+IgM(Zymed Laboratories, Inc., South San Francisco, Calif.) or goatanti-human Igs (Tago, Inc., Burlingame, Calif.) in bicarbonate buffer,pH 9.6. Plates were blocked for 2 hours at room temperature with 1% BSAin Tris-buffered saline, pH 7.5 (TBS), and then incubated at 37° C. for1 hour with standards or samples serially-diluted in TBS/1% BSA/0.05%Tween 20. Standards used were a monoclonal mouse IgG2a (IND1anti-melanoma; XOMA Corporation, Berkeley, Calif.) and polyclonal humanIg (Sigma Chemical Co., St. Louis, Mo.). Subsequently, plates werewashed with TBS/Tween 20 and incubated at 37° C. for 1 hour withalkaline phosphatase-conjugated goat anti-mouse IgG+IgA+IgM or goatanti-human Igs (Caltag Laboratories, South San Francisco, Calif.).Detection was by measurement of absorbance at 405 nm followingincubation with 1 mg/ml p-nitrophenylphosphate (Sigma) in 10%diethanolamine buffer, pH 9.8. Plasma from a normal BALB/c mouse wasused as a positive control in the mouse Ig ELISA. Plasma samples fromnaive SCID mice or normal BALB/c mice did not have detectable levels ofhuman Ig. Human slL-2R was quantified using an ELISA kit (ImmunotechS.A., Marseille, France) as per the manufacturer's instructions.

Five-to-seven week old mice with low plasma levels of mouse Ig (<10μg/ml) were preconditioned with an i.p. injection of cyclophosphamide(Sigma) at 200 mg/kg. Two days later, they were injected i.p. with25-40×106 freshly-isolated human PBL suspended in 0.8 ml PBS.

Immunoconjugate Treatment

SCID mice were bled at approximately 2 weeks after human PBLtransplantation. Mice with undetectable <10 pM or low plasma levels ofhuman slL-2R were eliminated from the study. The cut-off for exclusionof mice with detectable, but low, levels of human slL-2R was empiricallydetermined for each study and was generally 20 pM. The remaining micewere divided into groups and were administered vehicle orimmunoconjugate as an i.v. bolus (0.2 mg/kg) daily for 5 consecutivedays. Animals were sacrificed 1 day after cessation of treatment forquantitation of human T cells in tissues and human slL-2R in plasma.

Collection of Tissues and Analysis of PBL Depletion

Blood was collected from the retro-orbital sinus into heparinized tubes.Mice were then killed by cervical dislocation and spleens were removedaseptically. Single cell suspensions of splenocytes were prepared inHBSS by pressing the spleens between the frosted ends of sterile glassmicroscope slides. Collected cells were washed twice with PBS.Erythrocytes were eliminated from blood and splenocyte suspensions usingRBC lysing buffer. Subsequently, cells were resuspended in PBS forenumeration. Recovered cells were then assayed for Ag expression usingflow cytometry.

Two to five hundred thousand cells in 100 μl of PBS/1% BSA/0.1% sodiumazide were incubated on ice for 30 min. with saturating amounts ofvarious FITC- or phycoerythrin (PE)-conjugated Abs (Becton-Dickinson,Mountain View, Calif.) Abs used for staining included: HLe-1-FITC (IgG1anti-CD45), Leu 2-FITC (IgG1 anti-CD8), Leu 3 PE (IgG1 anti-CD4), andLeu M3-PE (IgG2a anti-CD14). Cells were then washed in cold buffer andfixed in 0.37% formaldehyde in PBS. Samples were analyzed on a FACscan(Becton-Dickinson) using log amplifiers. Regions to quantify positivecells were set based on staining of cells obtained from naive SCID mice.The absolute numbers of human Ag-positive cells recovered from SCIDtissues were determined by multiplying the percent positive cells by thetotal number of cells recovered from each tissue sample. The totalnumber of leukocytes in blood was calculated using a theoretical bloodvolume of 1.4 ml/mouse. The detection limit for accurate quantitation ofhuman cells in SCID mouse tissues was 0.05 %. All statistical comparisonbetween treatment groups were made using the Mann-Whitney U test.Treatment groups were determined to be significantly different frombuffer control groups when the p value was <0.05. Results are presentedin Table 10 below, wherein +indicates a significant difference fromcontrols, --indicates an insignificant difference and NT means theconjugate was not tested. CD5 Plus (XOMA Corporation, Berkeley, Calif.)is mouse H65 antibody chemically linked to RTA and is a positivecontrol. 0X19 Fab-Gel_(C247) is a negative control immunoconjugate. The0×19 antibody (European Collection of Animal Cell Cultures #84112012) isa mouse anti-rat CD5 antibody that does not cross react with human CD5.

                  TABLE 10                                                        ______________________________________                                                       Human T Cell Depletion                                         Test Article     Spleen     Blood                                             ______________________________________                                        CD5 Plus         +          +                                                 cH65 F(ab').sub.2                                                                              -          -                                                 cH65 Fab'        -          -                                                 H65-rGEL         +          +                                                 cH65 F(ab').sub.2 -rGel                                                                        +          +                                                 cH65 Fab'-rGel   +          +                                                 cH65 F(ab').sub.2 -Gel.sub.c247                                                                +          NT                                                cH65 Fab'-Gel.sub.c247                                                                         +          +                                                 he1H65 Fab'-Gel.sub.c247                                                                       +          NT                                                cH65 Fab'-Gel.sub.c44                                                                          +          +                                                 OX19 Fab-Gel.sub.c247                                                                          -          -                                                 ______________________________________                                    

All the gelonin immunoconj ugates were capable of depleting human cellsin the mouse model.

EXAMPLE 10

Nine genetic constructs were assembled that each included a naturalsequence gelonin gene fused to an H65 truncated heavy chain gene (Fd) oran H65 light chain gene (kappa). The H65 Fd sequence consists of thenucleotides encoding the murine H65 heavy chain variable (V), joining(J) and human IgG₁, constant (C) domain 1 regions. The DNA sequences ofthe V and J regions of the H65 Fd and kappa fragment genes linked to thepel B leader can be obtained from GenBank (Los Alamos NationalLaboratories, Los Alamos, N.M.) under Accession Nos. M90468 and M90467,respectively. Four of the gene fusions included a gelonin gene linked atthe 5' end of an H65 Fab fragment gene while the other four included angelonin gene linked at the 3' end of an H65 Fab fragment gene. A DNAlinker encoding a peptide segment of the E. coli shiga-like toxin (SLT)(SEQ ID NO: 58), which contains two cysteine residues participating in adisulfide bond and forming a loop that includes a protease sensitiveamino acid sequence) or of rabbit muscle aldolase (RMA) (SEQ ID NO: 59),which contains several potential cathepsin cleavage sites) was insertedbetween the gelonin gene and the antibody gene in the constructs.Alternatively, a direct fusion was made between a gelonin gene and anH65 Fab fragment gene without a peptide linker segment. Table 11 belowsets out a descriptive name of each gene fusion and indicates theexpression plasmid containing the gene fusion. Each plasmid alsoincludes the Fab fragment gene (shown in parentheses in Table 11 ) withwhich each particular gene fusion was co-expressed.

                  TABLE 11                                                        ______________________________________                                        Plasmid        Description                                                    ______________________________________                                        pING3754       Gelonin::SLT::Fd (kappa)                                       pING3757       Gelonin::SLT::kappa (Fd)                                       pING3759       Gelonin::RMA::Fd (kappa)                                       pING3758       Gelonin::RMA::kappa (Fd)                                       pING4406       Fd::SLT::Gelonin (kappa)                                       pING4407       kappa::SLT::Gelonin (Fd)                                       pING4408       Fd::RMA::Gelonin (kappa)                                       pING4410       kappa::RMA::Gelonin (Fd)                                       pING3334       Gelonin::Fd (kappa)                                            ______________________________________                                    

Fusions of Gelonin at the Carboxyl-Terminus of Antibody Genes

(1) Fd::SLT::Gelonin (kappa)

A gelonin gene fusion to the 3'-end of the H65 Fd chain with the 23amino acid SLT linker sequence was assembled in a three piece ligationfrom plasmids pVK1, pING3731 (ATCC 68721) and pING4000. Plasmid pVK1contains the Fd gene linked in-frame to the SLT linker sequence;pING3731 contains the gelonin gene, and pING4000 contains the H65 kappaand Fd genes each linked to the pelB leader sequence under the controlof the araB promoter as a dicistronic message.

Plasmid pVK1 was designed to link the 3'-end of a human IgG Fd₁ constantregion in-frame to a protease-sensitive segment of the SLT gene boundedby two cysteine residues which form an intra-chain disulfide bond. TheSLT gene segment (20 amino acids from SLT bounded by cysteine residues,plus three amino acids introduced to facilitate cloning) was assembledfrom two oligonucleotides, SLT Linker 1 and SLT Linker 2.

    ______________________________________                                        SLT Linker 1 (SEQ ID NO: 75)                                                  5' TGTCATCATCATGCATCGCGAGTTGCCAGAAT                                           GGCATCT                                                                       GATGAGTTTCCTTCTATGTGCGCAAGTACTC 3'                                            SLT Linker 2 (SEQ ID NO: 76)                                                  5' TCGAGAGTACTTGCGCACATAGAAGGAAACTCAT                                         CAGAT                                                                         GCCATTCTGGCAACTCGCGATGCATGATGAT                                               GACATGCA 3'                                                                   ______________________________________                                    

The two oligonucleotides were annealed and ligated into a vectorcontaining PstI and XhoI cohesive ends, destroying the PstI site andmaintaining the XhoI site. The vector, pING3185, contained an engineeredPst I site at the 3'-end of the Fd gene, and contained an XhoI sitedownstream of the Fd gene. The product of this ligation, pVK1, containedthe H65 Fd gene (fused to the pelB leader) in frame with the SLT linkersegment, and contained two restriction sites, FspI and ScaI, at the3'-end of the SLT linker.

Plasmid pVK1 was digested with SauI and ScaI, and the 217 bp fragmentcontaining a portion of the Fd constant domain and the entire SLT genesegment was purified by electrophoresis on an agarose gel. pING3731 wasdigested with SinI and XhoI and the 760 bp gelonin gene was similarlypurified. Plasmid pING4000 was digested with SauI and XhoI and thevector segment containing the entire kappa gene and a portion of the Fdgene was also purified. Ligation of these three DNA fragments resultedin pING4406 containing the Fd::SLT::Gelonin (kappa) gene fusion vector.

(2) kappa::SLT::Gelonin (Fd)

A gelonin gene fusion to the 3'-end of the H65 kappa chain with the 25amino acid SLT linker sequence (20 amino acids from SLT bounded bycysteine residues, plus 5 amino acids introduced to facilitate cloning)was assembled from the DNA segments in pING3731 (ATCC 68721) andpING3713.

Plasmid pING3713 is an Fab expression vector where the H65 Fd and kappagenes are linked in a dicistronic transcription unit containing the SLTlinker segment cloned in-frame at the 3'-end of the kappa gene. Theplasmid was constructed as follows. In a source plamid containing theH65 Fd and kappa genes, an EagI site was positioned at the 3-end of thekappa gene by site directed mutagenesis without altering the encodedamino acid sequence. The SLT gene segment from pVK1 was amplified withprimers SLT-EagI-5' and SalI for in frame linkage to the EagI site atthe 3'-end of the kappa gene.

    ______________________________________                                        SLT-Eag-5' (SEQ ID NO: 77)                                                    5' TGTTCGGCCGCATGTCATCATCATGCATCG 3'                                          SalI (SEQ ID NO: 78)                                                          5' AGTCATGCCCCGCGC 3'                                                         ______________________________________                                    

The 140 bp PCR product was digested with EagI and XhoI, and the 75 bpfragment containing the SLT gene segment was cloned adjacent to the Fdand kappa genes in the source plasmid to generate pING3713.

For construction of gene fusion to gelonin, pING3713 was cut with ScaIand XhoI, and the vector fragment containing the Fd gene and kappa::SLTfusion was purified. pING3731 was digested with SmaI and XhoI and theDNA fragment containing the gelonin gene was also purified. The productof the ligation of these two fragments, pING4407, contains the Fd andkappa::SLT::gelonin genes.

(3) Fd::RMA::Gelonin (kappa)

A gelonin gene fusion to the 3 '-end of the H65 Fd chain with the 21amino acid RMA linker sequence (20 amino acids from RMA, plus 1 aminoacid introduced to facilitate cloning) was assembled in a three pieceligation from plasmids pSH4, pING3731 (ATCC 68721) and pING4000.

Plasmid pSH4 contains an Fd gene linked in frame to the RMA linkersequence. The RMA gene segment was linked to the 3'-end of Fd by overlapextension PCR as follows. The 3 '-end (constant region) of the Fd genewas amplified by PCR from a source plasmid with the primers KBA-γ2 andRMAG-1. Any Fd constant region may be used because constant regions ofall human IgG₁ antibodies are identical.

    ______________________________________                                        KBA-γ2 (SEQ ID NO: 79)                                                  5' TCCCGGCTGTCCTACAGT 3'                                                      RMAG-1 (SEQ ID NO: 80)                                                        5' TCCAGCCTGTCCAGATGGTGTGTGAGTTTTGT                                           CACAA 3'                                                                      ______________________________________                                    

The product of this reaction was mixed with primer RMA-76, whichannealed to the amplified product of the first reaction, and the mixturewas amplified with primers KBA-γ2 and RMAK-2.

    ______________________________________                                        RMA-76 (SEQ ID NO: 81)                                                        5' CTAACTCGAGAGTACTGTATGCATGGTTCGAGAT                                         GAACA                                                                         AAGATTCTGAGGCTGCAGCTCCAGCCTGTCCAGATGG 3'                                      RMAK-2 (SEQ ID NO: 82)                                                        5' CTAACTCGAGAGTACTGTAT 3'                                                    ______________________________________                                    

The PCR product contained a portion of the Fd constant region linkedin-frame to the RMA gene segment. The product also contained a ScaIrestriction site useful for in-frame fusion to a protein such asgelonin, and an XhoI site for subsequent cloning. This PCR product wascut with Saul and XhoI and ligated adjacent to the remainder of the Fdgene to generate pSH4.

For assembly of the gene fusion vector containing the Fd::RMA::Gelonin,kappa genes, pSH4 was cut with Saul and ScaI and the Fd::RMA segment waspurified. Plasmid pING3731 was cut with SmaI and XhoI and the 760 bp DNAfragment containing the gelonin gene was purified, and pING4000 was cutwith SauI and XhoI and the vector was purified. The product of theligation of these fragments, pING4408, contained the Fd::RMA::Geloninand kappa genes.

(4) kappa::RMA::Gelonin (Fd)

A gelonin gene fusion to the 3 '-end of the H65 kappa chain with the 21amino acid RMA linker sequence was assembled in a three piece ligationfrom plasmids pSH6, pING4408 (see the foregoing paragraph) and pING3713.

Plasmid pSH6 contains a kappa gene linked in-frame to the RMA linkersequence. The RMA gene segment was linked to the 3'-end of kappa byoverlap extension PCR as follows. The 3 '-end (constant region) of thekappa gene was amplified by PCR from a source plamid with the primersKBA-K2 and RMAK-1.

    ______________________________________                                        RMAK-1 (SEQ ID NO: 83)                                                        5' TCCAGCCTGTCCAGATGGACACTCTCCCCTGTTGAA 3'                                    KBA-K2 (SEQ ID NO: 84)                                                        5' GTACAGTGGAAGGTGGAT 3'                                                      ______________________________________                                    

The product of this reaction was mixed with primer RMA-76 (SEQ ID NO:81), which annealed to the amplified product of the first reaction, andthe mixture was amplified with primers KBA-K2 and RMAK-2 (SEQ ID NO:82). The PCR product contained a portion of the kappa constant regionlinked in-frame to the RMA gene segment. The product also contained aScaI restriction site useful for in-frame fusion to a protein such asgelonin, and an XhoI site for subsequent cloning. This PCR product wascut with SstI and XhoI and ligated adjacent to the remainder to thekappa gene to generate pSH6.

For assembly of the gene fusion vector containing thekappa::RMA::Gelonin and Fd genes, pSH6 was cut with HindIII and PstI andthe DNA fragment containing the kappa constant region and a portion ofthe RMA linker (the PstI RMA linker segment contains a PstI site)segment was purified. Plasmid pING4408 was cut with PstI and SalI andthe DNA fragment containing a segment of the RMA linker, the geloningene and a portion of the tetracycline resistance gene in the vectorsegment was purified. pING3713 was cut with SalI and HindIII and thevector was purified. The product of the ligation of these threefragments, pING4410, contained the kappa::RMA::Gelonin and Fd genes.

Fusions of gelonin at the amino-terminus of antibody genes

(1) Gelonin::SLT::Fd (kappa)

A gelonin gene fusion to the 5'-end of the H65 Fd chain with a 25 aminoacid SLT linker sequence (20 amino acids from SLT bounded by cystineresidues, plus five amino acids introduced to facilitate cloning) wasassembled in a three piece ligation from plasmids pING3748, pING3217,and a PCR fragment encoding the H65 gamma variable region (V_(H)) genesegment which is the variable region of the Fd gene in pING3217. PlasmidpING3748 Contains the gelonin gene linked in-frame to the SLT linkersequence, and pING3217 contains the H65 Fd and kappa genes in adicistronic transcription unit.

Plasmid pING3825 (see Example 2) was amplified with PCR primersgelo3'-Eag and gelo-9 to introduce an EagI restriction site at the3'-end of the gelonin gene by PCR mutagenesis.

    ______________________________________                                        gelo3'-Eag (SEQ ID NO: 85)                                                    5' CATGCGGCCGATTTAGGATCTTTATCGACGA 3'                                         ______________________________________                                    

The PCR product was cut with BclI and EagI and the 56 bp DNA fragmentwas purified. Plasmid pING3713 was cut with EagI and XhoI, and the 77 bpDNA fragment containing the SLT linker was purified. The 56 bp BclI toEagI fragment and the 77 bp EagI to XhoI fragment were ligated intopING3825 which had been digested with BclI and XhoI to generate pING3748which contains the gelonin gene linked in-frame to the SLT linkersequence.

For assembly of the gene fusion vector containing the Gelonin::SLT::Fdand kappa genes, the H65 V_(H) was amplified by PCR from pING3217 withprimers H65-G1 and H65-G2, and the product was treated with T4polymerase followed by digestion with NdeI.

    ______________________________________                                        H65-G1 (SEQ ID NO: 86)                                                        5' AACATCCAGTTGGTGCAGTCTG 3'                                                  H65-G2 (SEQ ID NO: 87)                                                        5' GAGGAGACGGTGACCGTGGT 3'                                                    ______________________________________                                    

The 176 bp fragment containing the 5'-end of the H65 heavy chainV-region was purified. Concurrently, pING3217 was digested with NdeI andXhoI, and the 1307 bp DNA fragment containing a portion of the Fd geneand all of the kappa gene was purified. The two fragments were ligatedto pING3748 which had been digested with ScaI and XhoI in a three pieceligation yielding pING3754 (ATCC 69102), which contains theGelonin::SLT::Fd and kappa genes.

(2) Gelonin::SLT::kappa (Fd)

A gelonin gene fusion to the 5'-end of the H65 kappa chain with the 25amino acid SLT linker sequence was assembled in a three piece ligationfrom plasmids pING3748 (see the foregoing section), pING4000, and a PCRfragment encoding the H65 kappa variable region (V_(L)) gene segment.

For assembly of the gene fusion vector containing theGelonin::SLT::kappa and Fd genes, an H65 V_(L) fragment was amplified byPCR from pING3217 with primers H65-K1 and JK1-HindIII, and the productwas treated with T4 polymerase followed by digestion with HindIII.

    ______________________________________                                        H65-K1 (SEQ ID NO: 88)                                                        5' GACATCAAGATGACCCAGT 3'                                                     JK1-HindIII (SEQ ID NO: 89)                                                   5' GTTTGATTTCAAGCTTGGTGC 3'                                                   ______________________________________                                    

The 306 bp fragment containing the light chain V-region was purified.Concurrently, pING4000 was digested with HindIII and XhoI, and the 1179bp DNA fragment containing the kappa constant region and all of the Fdgene was purified. The two fragments were ligated to pING3748 which hadbeen digested with ScaI and XhoI in a three piece ligation yieldingpING3757, which contains the Gelonin::SLT::kappa and Fd genes.

(3) Gelonin::RMA::Fd (kappa)

A gelonin gene fusion to the 5'-end of the H65 Fd chain with the 24amino acid RMA linker sequence (20 amino acids from RMA, plus 4 aminoacids introduced to facilitate cloning) was assembled in a three pieceligation from plasmids pING3755, pING3217, and a PCR fragment encodingthe H65 V_(H) gene segment. Plasmid pING3755 contains the gelonin genelinked in-frame to the RMA linker sequence, and pING3217 contains theH65 Fd and kappa genes in a dicistronic transcription unit.

Plasmid pING3755 was assembled to contain the gelonin gene linked to theRMA linker gene segment. The RMA linker gene segment was amplified byPCR from pSH4 with primers RMA-EagI and HINDIII-2.

    ______________________________________                                        RMA-EagI (SEQ ID NO: 90)                                                      5' ACTTCGGCCGCACCATCTGGACAGGCTGGAG 3'                                         HINDIII-2 (SEQ ID NO: 91)                                                     5' CGTTAGCAATTTAACTGTGAT 3'                                                   ______________________________________                                    

The 198 bp PCR product was cut with EagI and HindIII, and the resulting153 bp DNA fragment was purified. This RMA gene segment was clonedadjacent to gelonin using an PstI to EagI fragment from pING3748 and thePstI to HindIII vector fragment from pING3825. The product of this threepiece ligation was pING3755.

For assembly of the gene fusion vector containing the Gelonin::RMA::Fd,kappa genes, the H65 V_(H) was amplified by PCR from pING3217 withprimers H65-G1 (SEQ ID NO: 86) and H65-G2 (SEQ ID NO: 87), and theproduct was treated with T4 polymerase followed by digestion with NdeI.The 186 bp fragment containing the 5'-end of the heavy chain V-regionwas purified. Concurrently, pING3217 was digested with NdeI and XhoI,and the 1307 bp DNA fragment containing a portion of the Fd gene and allof the kappa gene was purified. These two fragments were ligated topING3755 which had been digested with ScaI and XhoI in a three pieceligation yielding pING3759 (ATCC 69104), which contains theGelonin::RMA::Fd and kappa genes.

(4) Gelonin::RMA::kappa (Fd)

A gelonin gene fusion to the 5'-end of the H65 kappa chain with the 24amino acid RMA linker sequence was assembled in a three piece ligationfrom plasmids pING3755, pING4000, and a PCR fragment encoding the H65V_(L) gene segment.

For assembly of the gene fusion vector containing theGelonin::RMA::kappa and Fd genes, an H65 V_(L) segment was amplified byPCR from pING3217 with primers H65K-1 (SEQ ID NO: 88) and JK1-HindIII(SEQ ID NO: 89), and the product was treated with T4 polymerase followedby digestion with HindIII. The 306 bp fragment containing the 5'-end ofthe light chain V-region was purified. Concurrently, pING4000 wasdigested with HindIII and XhoI, and the 1179 bp DNA fragment containingthe kappa constant region and all of the Fd gene was purified. These twofragments were ligated to pING3755 which had been digested with ScaI andXhoI in a three piece ligation yielding pING3758 (ATCC 69103), whichcontains the Gelonin::RMA::kappa and Fd genes.

(5) Gelonin::Fd (Kappa)

A direct gelonin gene fusion was constructed from pING3754. pING3754 wasdigested with BglII and XhoI and the vector segment was purified.Concurrently, pING3754 was digested with EagI, treated with T4polymerase, cut with BglII, and the gelonin gene segment was purified.pING3754 was also cut with FspI and XhoI, and the Fd and kappa genesegment was purified. These fragments were assembled in a three-pieceligation to generate pING3334, which contains a direct gene fusion ofgelonin to Fd in association with a kappa gene.

EXAMPLE 11

Each of the eight gelonin gene fusions whose construction is describedin Example 10 was co-expressed with its pair H65 Fab gene inarabinose-induced E. coli strain E104.

Expression products of the gene fusions were detected in the supernatantof induced cultures by ELISA. Typically, a plate was coated withantibody recognizing gelonin. Culture supernatant was applied and boundFab was detected with antibody recognizing human kappa coupled tohorseradish peroxidase. H65 Fab fragment chemically conjugated togelonin was used a standard. Alternative ELISA protocols involvingcoating a plate with antibody recognizing either the kappa or Fd orinvolving a detection step with anti-human Fd rather that anti-humankappa yielded similar results. Only properly assembled fusion proteincontaining gelonin, kappa and Fd was detected by this assay.Unassociated chains were not detected.

The fusion protein produced from induced cultures containing expressionvectors pING4406, 4407, 4408, and 4410 in E. coli E104 accumulated atabout 20-50 ng/ml. The fusion proteins expressed upon induction ofpING3754, 3334, 3758 and 3759 (but not pING3757) were expressed at muchhigher levels, at about 100 to 500 ng/ml. A fusion protein of about70,000 Kd was detected in the concentrated E. coli culture supernatantby immunostaining of Western blots with either anti-human kappa oranti-gelonin antibodies.

The Gelonin::SLT::Fd (kappa) fusion protein from pING3754 (ATCC 69102)was purified from induced 10 L fermentation broth. The 10 L fermentationbroth was concentrated and buffer exchanged into 10mM phosphate bufferat pH 7.0, using an S10Y10 cartridge (Amicon) and a DC10 concentrator.The supernatant was purified by passing the concentrated supernatantthrough a DE52 column (20×5 cm) equilibrated with 10 mM sodium phosphatebuffer at pH 7.0. The flow-through was then further purified andconcentrated by column chromatography on CM52 (5×10 cm) in 10 mMphosphate buffer. A 0-0.2 M linear gradient of NaCl was used to theelute the fusion protein, and fractions containing the fusion proteinwere pooled and loaded onto a Protein G column (1 ml). The fusionprotein was eluted from protein G with 0.2 M glycine. TheGelonin::RMA::Fd (kappa) and Gelonin::RMA::kappa (Fd) fusions proteinswere purified from fermentation broths by similar methods except thatthe CM52 column step was eliminated, and the DE52 column wasequilabrated with 100 mM sodium phosphate buffer at pH 7.0. The fusionproteins were not purified to homogeneity.

Each of the three purified fusion proteins was then assayed for activityin the RLA assay and for cytotoxicity against the T-cell line HSB2. (Tcells express the CD5 antigen which is recognized by H65 antibody.) TheRLA assay was performed as described in Example 4 and results of theassay are presented below in Table 12.

                  TABLE 12                                                        ______________________________________                                        Fusion Protein      IC50 (pM)                                                 ______________________________________                                        rGelonin            11                                                        Gelonin::SLT::Fd (kappa)                                                                          19                                                        Gelonin::RMA::Fd (kappa)                                                                          28                                                        Gelonin::RMA::kappa (Fd)                                                                          10                                                        ______________________________________                                    

In whole cell cytotoxicity assays performed as described in Example 6,the fusion protein was active and killed two T cell lines, HSB2 and CEM,with respective IC₅₀ s 2-fold (HSB2) or 10-fold (CEM) lower than that ofthe gelonin chemically linked to H65. See Table 13 below for resultswherein IC₅₀ values were adjusted relative to the amount of fusionprotein in each sample.

                  TABLE 13                                                        ______________________________________                                        IC.sub.50 (pMT)                                                               Fusion Protein     HSB2 Cells                                                                              CEM Cells                                        ______________________________________                                        he3Fab-Gel.sub.C44 165        173                                             Gelonin:SLT::Fd (kappa)                                                                          180       1007                                             Gelonin::RMA::Fd (kappa)                                                                         150       nt                                               ______________________________________                                    

The fusion protein showed similar activity on peripheral bloodmononuclear cells (dam not shown).

EXAMPLE 12

The natural sequence gelonin gene was also fused to a single chain formof the human engineered he3 H65 variable region. The gelonin gene waspositioned at the N-terminus of the fusion gene and the SLT or RMAlinker peptide was positioned between the gelonin and antibody domainsto allow intracellular processing of the fusion protein with subsequentcytosolic release of gelonin.

A single chain antibody (scAb) form of the he3 H65 variable domain wasassembled from previously constructed genes. This scab segment consistedof the entire V and J region of the one chain (heavy or light) linked tothe entire V and J segment of the other chain (heavy or light) via a 15amino acid flexible peptide: [(Gly)₄ Ser]₃. This peptide is identical tothat described in Huston et al., Proc. Natl. Acad. Sci. USA, 85,5879-5883 (1988); Glockshuber et al., Biochemistry, 29, 1362-1367(1990); and Cheadle et al., Molecular Immunol., 29, 21-30 (1992). ThescAb was assembled in two orientations: V-J_(kappa) ::[(Gly)₄ Ser]₃::V-J_(Gamma) (SEQ ID NO: 92) and V-J_(Gamma) ::[(Gly)₄ Ser]₃::V-J_(kappa) (SEQ ID NO: 93). Each scab segment was assembled andsubsequently fused to gelonin.

For assembly of the scAb segment V-J_(kappa) ::[(Gly)₄Ser]3::V-J_(Gamma), primers HUK-7 and SCFV-1 were used to amplify a 352bp DNA fragment containing the he3 V/J kappa sequences from pING4627 byPCR in a reaction containing 10 mM KCl, 20 mM TRIS pH 8.8, 10 mM (NH4)₂SO₂, 2 mM MgSO₄, 0.1% Triton X-100., 100 ng/ml BSA, 200 uM of each dNTP,and 2 Units of Vent polymerase (New England Biolabs, Beverley, Mass.) ina total volume of 100 μl.

    ______________________________________                                        SCFV-1 (SEQ ID NO: 94)                                                        5' CGGACCCACCTCCACCAGATCCACCGC                                                CACCTTTCATCTCAAGCTTGGTGC 3'                                                   HUK-7 (SEQ ID NO: 95)                                                         5' GACATCCAGATGACTCAGT 3'                                                     ______________________________________                                    

Concurrently, primers SCFV-2 and SCFV-3 were used to amplify a he3 heavychain V/J gamma segment from pING4623, generating a 400 bp fragment.

    ______________________________________                                        SCFV-2 (SEQ ID NO: 96)                                                        5' GGTGGAGGTGGGTCCGGAGGTGGAGGATCTGA                                           GATCCAGTTGGTGCAGT 3'                                                          SCFV-3 (SEQ ID NO: 97)                                                        5'TGTACTCGAGCCCATCATGAGGAGACGGTGACCGT3'                                       ______________________________________                                    

The products from these reactions were mixed and amplified with theoutside primers HUK-7 and SCFV-3. The product of this reaction wastreated with T4 polymerase and then cut with XhoI. The resulting 728 bpfragment was then purified by electrophoresis on an agarose gel. Thisfragment was ligated into the vectors pING3755 and pING3748 (see Example10), each digested with ScaI and XhoI. The resulting vectors pING4637and pING4412 contain the Gelonin::RMA::scab V-J_(kappa) ::[(Gly)₄ Ser]₃::V-J_(Gamma) and Gelonin::SLT::scAb V-J_(kappa) ::[(Gly)₄ Ser]₃::V-J_(Gamma) fusion genes, respectively.

Similarly, the scAb V-J_(Gamma) ::[(Gly)₄ Ser]₃ ::V-J_(kappa) wasassembled by amplification of pING4627 with primers SCFV-5 and SCFV-6generating a 367 bp fragment containing he3 V/J kappa sequences,

    ______________________________________                                        SCFV-5 (SEQ ID NO: 98)                                                        5' GGTGGAGGTGGGTCCGGAGGTGGAGGATCT 3'                                          SCFV-6 (SEQ ID NO: 99)                                                        5' TGTACTCGAGCCCATCATTTCATCTCAAGCTTGGTGC 3'                                   ______________________________________                                    

and pING4623 with primers H65-G3 and SCFV-4 generating a 385 bp fragmentcontaining he3 gamma V/J sequences by PCR with Vent polymerase.

    ______________________________________                                        H65-G3 (SEQ ID NO: 100)                                                       5' GAGATCCAGTTGGTGCAGTCTG 3'                                                  SCFV-4 (SEQ ID NO: 101)                                                       5' CGGACCCACCTCCACCAGATCC                                                     ACCGCCACCTGAGGAGACGGTGACCGT 3'                                                ______________________________________                                    

The products from these reactions were mixed and amplified with H65-G3and SCFV-6. The 737 bp product was treated with T4 polymerase and cutwith XhoI. Ligation into pING3755 and pING3748 (digested with ScaI andXhoI) resulted in assembly of the Gelonin::RMA::scAb V-J_(Gamma)::[(Gly)₄ Ser]₃ ::V-J_(kappa) gene fusion in pING4638 andGelonin::SLT::scAb V-J_(Gamma) ::[(Gly)₄ Ser]₃ ::V-J_(kappa) gene fusionin pING4639, respectively.

The vectors pING4637, pING4412, pING4638 and pING4639 were eachtransformed into E. coli strain E104 and induced with arabinose. Proteinproducts of the predicted molecular weight were identified by Westernblot with gelonin-specific antibodies.

Gelonin::scAb fusions without a cleavable linker can be constructed bydeletion of the SLT linker in pING4412 using the restriction enzymesEagI and FspI. Digestion at these sites and religation of the plasmidresults in an in-frame deletion of the SLT sequence.

EXAMPLE 13

BRIP possesses characteristics which make it an attractive candidate fora component of immunotoxins. BRIP is a naturally unglycosylated proteinthat may have reduced uptake in the liver and enhanced circulatoryresidence time in vivo. Additionally, BRIP is less toxic and lessimmunogenic in animals than the A-chain of ricin. Cloning of the BRIPgene and expression of recombinant BRIP in an E. coli expression systemobviates the need to purify native BRIP directly from barley, andenables the development of analogs of BRIP which may be conjugated withan available cysteine residue for conjugation to antibodies.

Purification of BRIP and Generation of Polyclonal Antibodies to BRIP

Native BRIP was purified from pearled barley flour. Four kilograms offlour was extracted with 16 liters of extraction buffer (10 mM NaPO4, 25mM NaCl, pH 7.2) for 20 hours at 4° C. The sediment was removed bycentrifugation, and 200 ml of packed S-Sepharose (Pharmacia, Piscataway,N.J.) was added to absorb BRIP. After mixing for 20 hours at 4° C., theresin was allowed to settle out, rinsed several times with extractionbuffer and then packed into a 2.6×40 cm column. Once packed, the columnwas washed with extraction buffer (150 ml/h) until the absorbance of theeffluent approached zero. BRIP was then eluted with a linear gradient of0.025 to 0.3 M NaCl in extraction buffer and 5 ml fractions werecollected. BRIP-containing peaks (identified by Western analysis ofcolumn fractions) were pooled, concentrated to about 20 ml, and thenchromatographed on a 2.6×100 cm Sephacryl S-200HR (Pharmacia) columnequilibrated in 10 mM NaPO₄, 125 mM NaCl, pH 7.4 (10 ml/hr).BRIP-containing peaks were pooled again, concentrated, and stored at-70° C.

The resulting purified BRIP protein had a molecular weight of about30,000 Daltons, based upon the mobility of Coomassie-stained proteinbands following SDS-PAGE. The amino acid composition was consistent withthat published by Asano et al., Carlsberg Res. Comm., 49, 619-626(1984).

Rabbits were immunized with purified BRIP to generate polyclonalantisera.

Cloning of the BRIP Gene

A cDNA expression library prepared from germinating barley seeds in thephage λ expression vector λZAPII was purchased from Stratagene, LaJolla, Calif. Approximately 700,000 phage plaques were screened withanti-BRIP polyclonal antisera and 6 immunoreactive plaques wereidentified. One plaque was chosen, and the cDNA contained therein wasexcised from λZAPII with EcoRI and subcloned into pUC18 generating thevector pBS1. The cDNA insert was sequenced with Sequenase (United StatesBiochemical, Cleveland, Ohio). The DNA sequence of the native BRIP geneis set out in SEQ ID NO: 12. To confirm that cDNA encoded the nativeBRIP gene, the cDNA was expressed in the E. coli plasmid pKK233-2(Pharmacia). BRIP protein was detected in IPTG-induced cells transformedwith the plasmid by Western analysis with above-described rabbitanti-BRIP antisera.

Construction of an E. coli Expression Vector Containing the BRIP Gene

Barley cDNA containing the BRIP gene was linked to a pelB leadersequence and placed under control of an arab promoter in a bacterialsecretion vector.

An intermediate vector containing the BRIP gene linked to the pelBleader sequence was generated. Plasmid pBS1 was cut with NcoI, treatedwith Mung Bean Nuclease, cut with BamHI and the 760 bp fragmentcorresponding to amino acids 1-256 of BRIP was purified from an agarosegel. Concurrently, a unique XhoI site was introduced downstream of the3'-end of the BRIP gene in pBS1 by PCR amplification with a pUC18 vectorprimer (identical to the Reverse® primer sold by NEB or BRL butsynthesized on a Cyclone Model 8400 DNA synthesizer) and the specificprimer BRIP 3'Xho. The sequence of each of the primers is set out below.

    ______________________________________                                        Reverse (SEQ ID NO: 45)                                                       5' AACAGCTATGACCATG 3'                                                        BRIP 3'Xho (SEQ ID NO: 46)                                                    5' TGAACTCGAGGAAAACTACCTATTTCCCAC 3'                                          ______________________________________                                    

Primer BRIP 3'Xho includes a portion corresponding to the last 8 bp ofthe BRIP gene, the termination codon and several base pairs downstreamof the BRIP gene, and an additional portion that introduces a XhoI sitein the resulting PCR fragment. The PCR reaction product was digestedwith BamHI and XhoI, and an 87 bp fragment containing the 3'-end of theBRIP gene was purified on a 5% acrylamide gel. The 760 and 87 bppurified BRIP fragments were ligated in the vector pING 1500 adjacent tothe pelB leader sequence. pING1500 had previously been cut with SstI,treated with T4 polymerase, cut with XhoI, and purified. The DNAsequence at the junction of the pelB leader and the 5'-end of the BRIPgene was verified by DNA sequence analysis. This vector was denotedpING3321-1.

The final expression vector was assembled by placing the BRIP gene underthe control of the inducible araB promoter. Plasmid pING3321-1 was cutwith PstI and XhoI, and the BRIP gene linked to the pelB leader waspurified from an agarose gel. The expression vector pING3217, containingthe arab promoter, was cut with PstI and XhoI and ligated to the BRIPgene. The expression vector was denoted pING3322.

Arabinose induction of E. coli cells containing the plasmid pING3322 ina fermenter resulted in the production of about 100 mg per liter ofrecombinant BRIP. E. coli-produced BRIP displays properties identical toBRIP purified directly from barley seeds.

Construction of BRIP Analogs With a Free Cysteine Residue

The BRIP protein contains no cysteine residues, and therefore containsno residues directly available which may form a disulfide linkage toantibodies or other proteins. Analogs of recombinant BRIP were generatedwhich contain a free cysteine residue near the C-terminus of theprotein. Three residues of the BRIP protein were targets for amino acidsubstitutions. Comparison of the amino acid sequence of BRIP to theknown tertiary structure of the ricin A-chain (see FIG. 2) suggestedthat the three positions would be available near the surface of themolecule. The three BRIP analogs include cysteines substituted in placeof serine₂₇₇, alanine₂₇₀, and leucine₂₅₆ of the native protein, and weredesignated BRIP_(C277), BRIP_(C270) and BRIP_(C256), respectively.

(1) A plasmid vector capable of expressing the BRIP_(C277) analog wasconstructed by replacing the 3'-end of the BRIP gene with a DNA segmentconferring the amino acid change. The EcoRI fragment containing the BRIPgene from pBS1 was subcloned into M13mp18, and single-stranded DNA(anti-sense strand) was amplified by PCR with primers OBM2(corresponding nucleotides -11 to +8 of the BRIP gene) and OMB4(corresponding to amino acids 264-280 of BRIP and the termination codonof BRIP, and incorporating the substitution of a cysteine codon for thenative codon for serine₂₇₇ of native BRIP). The sequences of primersOBM2 and OMB4, wherein the underlined nucleotides encode the substitutedcysteine, are set out below.

    ______________________________________                                        OBM2 (SEQ ID NO: 47)                                                          5' GCATTACATCCATGGCGGC 3'                                                     OMB4 (SEQ ID NO: 48)                                                           ##STR27##                                                                    CATGGAACAGCTCCAGCGCCTTGGCCACCGTC 3'                                           ______________________________________                                    

A fragment containing a BRIP gene in which the codon for the amino acidat position 277 was changed to a cysteine codon was amplified. Thefragment was cloned into the SmaI site of pUC19 (BRL) and the plasmidgenerated was denoted pMB22. pMB22 was digested with EcoRI and anEcoRI-XhoI linker (Clonetech, Palo Alto, Calif.) was ligated into thevector. Subsequent digestion with XhoI and religation generated vectorpINGMB2X. A BamHI to XhoI fragment encoding the 3'-end of BRIP with thealtered amino acid was excised from pMB2X and the fragment was purifiedon a 5% acrylamide gel. This fragment along with an EcoRI to BamHIfragment containing the pelB leader sequence and sequences encoding thefirst 256 amino acids of BRIP were substituted in a three piece ligationinto pING3322 cut with EcoRI and XhoI. The resulting vector containingthe BRIP_(C277) analog was designated pING3803 (ATCC Accession No.68722).

(2) A BRIP analog with a free cysteine at position 256 was constructedusing PCR to introduce the amino acid substitution. A portion of theexpression plasmid pING3322 was amplified with primers BRIP-256 andHINDIII-2. The sequence of each primer is set out below.

    ______________________________________                                        BRIP-256 (SEQ ID NO: 49)                                                       ##STR28##                                                                    HINDIII-2 (SEQ ID NO: 50)                                                     5' CGTTAGCAATTTAACTGTGAT 3'                                                   ______________________________________                                    

Nucleotides 4-21 of primer BRIP-256 encode amino acids 256-262 of BRIPwhile the underlined nucleotides specify the cysteine to be substitutedfor the leucine at the corresponding position of the native BRIPprotein. Primer HINDIII-2 corresponds to a portion of the plasmid. ThePCR product, which encodes the carboxyl terminal portion of the BRIPanalog, was treated with T4 polymerase, cut with XhoI, and the resultingfragment was purified on a 5% acrylamide gel. Concurrently, plasmidpING3322 was cut with BamHI, treated with T4 polymerase, cut with EcoRI,and the fragment containing the pelB leader sequence and sequencesencoding the first 256 amino acids of BRIP was purified. The twofragments were then assembled back into pING3322 to generate the geneencoding the analog BRIP_(C256). This plasmid is denoted pING3801.

(3) A BRIP analog with a cysteine at position 270 was also generatedusing PCR. A portion of the expression plasmid pING3322 was amplifiedwith primers BRIP-270 and the HINDIII-2 primer (SEQ ID NO: 50). Thesequence of primer BRIP-270 is set out below.

    ______________________________________                                        BRIP-270 (SEQ ID NO: 51)                                                      5' CCAAGTGTCTGGAGCTGTTCCATGCGA 3'                                             ______________________________________                                    

Primer BRIP-270 corresponds to amino acids 268-276 of BRIP with theexception of residue 270. The codon of the primer corresponding toposition 270 specifies a cysteine instead of the alanine present in thecorresponding position in native BRIP. The PCR product was treated withT4 polymerase, cut with XhoI, and the 51 bp fragment, which encodes thecarboxyl terminal portion of the analog, was purified on a 5% acrylamidegel. The fragment (corresponding to amino acids 268-276 of BRIP_(C270))was cloned in a three piece ligation along with the internal 151 bp BRIPrestriction fragment from SstII to MscI (corresponding to BRIP aminoacids 217-267) from plasmid pING3322, and restriction fragment fromSstII to XhoI from pING3322 containing the remainder of the BRIP gene.The plasmid generated contains the gene encoding the BRIP_(C270) analogand is designated pING3802.

Purification of Recombinant BRIP and the BRIP Analogs

Recombinant BRIP (rBRIP) and the BRIP analogs with free cysteineresidues were purified essentially as described for native BRIP exceptthey were prepared from concentrated fermentation broths. For rBRIP,concentrated broth from a 10 liter fermentation batch was exchanged into10 mM Tris, 20 mM NaCl pH 7.5, loaded onto a Sephacryl S-200 column, andeluted with a 20 to 500 mM NaCl linear gradient. Pooled rBRIP wasfurther purified on a Blue Toyopearl® column (TosoHaas) loaded in 20 mMNaCl and eluted in a 20 to 500 mM NaCl gradient in 10mM Tris, pH 7.5.For BRIP analogs, concentrated fermentation broths were loaded onto aCM52 column (Whatman) in 10 mM phosphate buffer, pH 7.5, and eluted witha 0 to 0.3M NaCl linear gradient. Further purification was bychromatography on a Blue Toyopearl® column.

Reticulocyte Lysate Assay

The ability of the rBRIP and the BRIP analogs to inhibit proteinsynthesis in vitro was tested by reticulocyte lysate assay as describedin Example 1. Serial log dilutions of standard toxin (RTA 30), nativeBRIP, rBRIP and BRIP analogs were tested over a range of 1 μg/ml to 1pg/ml. By comparison with an uninhibited sample, the picomolarconcentration of toxin (pM) which corresponds to 50% inhibition ofprotein synthesis (IC₅₀) was calculated. The results of the assays arepresented below in Table 14.

                  TABLE 14                                                        ______________________________________                                        Toxin          IC.sub.50 (pM)                                                 ______________________________________                                        RTA 30         3.1                                                            Native BRIP    15                                                             rBRIP          18                                                             BRIP.sub.C256  23                                                             BRIP.sub.C270  20                                                             BRIP.sub.C277  24                                                             ______________________________________                                    

The RLA results indicate that the BRIP analogs exhibitribosome-inactivating activity comparable to that of the recombinant andnative BRIP toxin. All the analogs retained the natural ability ofnative BRIP to inhibit protein synthesis, suggesting that amino acidsubstitution at these positions does not affect protein folding andactivity.

Construction of BRIP Immunoconjugates

Immunoconjugates of native BRIP with 4A2 (described in Morishima et al.,J. Immunol., 129, 1091 (1982) and H65 antibody (obtained from hybridomaATCC HB9286) which recognize the T-cell determinants CD7 and CD5,respectively, were constructed. Immunoconjugates of ricin A-chains(RTAs) with 4A2 and H65 antibody were constructed as controls. The H65antibody and ricin A-chains as well as the RTA immunoconjugates wereprepared and purified according to methods described in U.S. patentapplication Ser. No. 07/306,433 supra and in International PublicationNo. WO 89/06968.

To prepare immunoconjugates of native BRIP, both the antibody (4A2 orH65) and native BRIP were chemically modified with the hindered linker5-methyl-2-iminothiolane (M2IT) at lysine residues to introduce areactive sulfhydryl group as described in Goff et al., BioconjugateChem., 1, 381-386 (1990). BRIP (3 mg/ml) was first incubated with 0.5 mMM2IT and 1 mM DTNB in 25 mM triethanolamine, 150 mM NaCl, pH 8.0, for 3hours at 25° C. The derivitized BRIP-(M2IT)-S-S-TNB was then desalted ona column of Sephadex GF-05LS and the number of thiol groups introducedwas quantitated by the addition of 0.1 mM DTT. On average, each BRIPmolecule contained 0.7 SH/mol.

4A2 or H65 antibody (4 mg/ml) in triethanolamine buffer was similarlyincubated with M2IT (0.3 mM) and DTNB (1 mM) for 3 hours at 25° C.Antibody-(M2IT)-S-S-TNB was then desalted and the TNB:antibody ratio wasdetermined. To prepare the conjugate, the BRIP-(M2IT)-S-S-TNB was firstreduced to BRIP-(M2IT)-SH by treatment with 0.5 mM DTT for 1 hour at 25°C., desalted by gel filtration of Sephadex® GF-05LS to remove thereducing agent, and then mixed with antibody-(M2IT)-S-S-TNB.

Following a 3 hour incubation at 25° C., and an additional 18 hours at4° C., the conjugate was purified by sequential chromatography on AcA44(IBF) and Blue Toyopearl®. Samples of the final product were run on 5%non-reducing SDS PAGE, Coomassie stained, and scanned with a Shimadzulaser densitometer to quantitate the number of toxins per antibody.

The BRIP analogs containing a free cysteine were also conjugated to 4A2and H65 antibodies. The analogs were treated with 50 mM DTT either for 2hours at 25° C. or for 18 hours at 4° C. to expose the reactivesulfhydryl group of the cysteine and desalted. The presence of a freesulfhydryl was verified by reaction with DTNB [Ellman et al., Arch.Biochem. Biophys, 82, 70-77 (1959)]. 4A2 or H65 antibody derivatized asdescribed above with M2IT was incubated with the reduced BRIP analogs ata ratio of 1:5 at room temperature for 3 hours and then overnight at 4°C. Immunoconjugates H65-BRIP_(C256), 4A2-BRIP_(C256), H65-BRIP_(C277)were prepared in 25 mM triethanolamine, 150 mM NaCl pH 8, whileimmunoconjugates H65-BRIP₂₇₀, 4A2-BRIP_(C270) and 4A2-BRIP_(C277) wereprepared in 0.1 M sodium phosphate, 150 mM NaCl pH 7.5. Followingconjugation, 10 μM mercaptoethylamine was added for 15 minutes at 25° C.to quenched any unreacted m2IT linkers on the antibody. The quenchedreaction solution was promptly loaded onto a gel filtration column(AcA44) to remove unconjugated ribosome-inactivating protein.Purification was completed using soft gel affinity chromatography onBlue Toyopearl® resin using a method similar to Knowles et al., Analyt.Biochem., 160, 440 (1987). Samples of the final product were run on 5%non-reduced SDS PAGE, Coomassie stained, and scanned with a Shimadzulaser densitometer to quantitate the number of toxins per antibody. Theconjugation efficiency was substantially greater for BRIP_(C277) (78%)than for either of the other two analogs, BRIP_(C270) and BRIP_(C256)(each Of these was about 10%). Additionally, the BRIP_(C277) product wasa polyconjugate, i.e., several BRIP molecules conjugated to a singleantibody, in contrast to the BRIP_(C270) and BRIP_(C256) products whichwere monoconjugates.

Whole Cell Kill Assay

Immunoconjugates of native BRIP and of the BRIP analogs were tested forthe ability to inhibit protein synthesis in HSB2 cells by the whole cellkill assay described in Example 1. Standard immunoconjugates H65-RTA(H65 derivatized with SPDP linked to RTA) and 4MRTA (4A2 antibodyderivatized with M2IT linked to RTA) and BRIP immunoconjugate sampleswere diluted with RPMI without leucine at half-log concentrationsranging from 2000 to 0.632 ng/ml. All dilutions were added in triplicateto microtiter plates containing 1×10⁵ HSB2 cells. HSB2 plates wereincubated for 20 hours at 37° C. and then pulsed with ³ H-Leu for 4hours before harvesting. Samples were counted on the Inotec Trace 96cascade ionization counter. By comparison with an untreated sample, thepicomolar toxin concentration (pM T) of immunoconjugate which resultedin a 50% inhibition of protein synthesis (IC₅₀) was calculated. Theassay results are presented below in Table 15.

                  TABLE 15                                                        ______________________________________                                        Conjugate      IC.sub.50 (pM T)                                               ______________________________________                                        4A2-BRIP       122.45                                                         4A2-BRIP.sub.C270                                                                            46.3                                                           4A2-BRIP.sub.C277                                                                            57.5                                                           4A2-BRIP.sub.C256                                                                            1116                                                           H65-BRIP       >5000                                                          H65-BRIP.sub.C277                                                                            1176                                                           ______________________________________                                    

The BRIP analog conjugates were less potent than the ricin conjugatecontrol (dam not shown). The immunotoxins containing antibody 4A2 andeither the BRIP_(C270) or the BRIP_(C277) analog exhibited comparable toincreased specific cytotoxicity toward target cells as compared toimmunotoxin containing native BRIP. While 4A2-BRIP_(C256) is less activethan 4A2-BRIP, 4A2-BRIP_(C270) and 4A2-BRIP_(C277) are between 3 and 4times more active. Similarly, the immunoconjugate of H65 to BRIP_(C277)shows greater toxicity toward target cells than the immunoconjugate ofH65 to native BRIP. Thus, linkage of antibody to BRIP derivatives whichhave an available cysteine residue in an appropriate location results inimmunotoxins with enhanced specific toxicity toward target cellsrelative to conjugates with native BRIP.

Disulfide Bond Stability Assay

Immunoconjugates prepared with native BRIP and the BRIP analogs wereexamined by the disulfide bond stability assay described in Example 1.Briefly, conjugates were incubated with increasing concentrations ofglutathione for 1 hour at 37° C. and, after terminating the reactionwith iodoacetamide, the amount of RIP released was quantitated bysize-exclusion HPLC on a TosoHaas TSK-G2000SW column.

By comparisons with the amount of RIP released by high concentrations of2-mercaptoethanol (to determine 100% release), the concentration ofglutathione required to release 50% of the RIP (the RC₅₀) wascalculated. As shown below in Table 16, the conjugates prepared withBRIP_(C270) or BRIP_(C277) were significantly more stable than eitherthe RTA conjugates or those prepared with native BRIP.

                  TABLE 16                                                        ______________________________________                                        Conjugate       RC.sub.50 (mM)                                                ______________________________________                                        H65-RTA         7.0                                                           H65-BRIP        2.8                                                           H65-BRIPC277    196.0                                                         4A2-RTA         4.4                                                           4A2-BRIP        3.3                                                           4A2-BRIP.sub.C270                                                                             53.0                                                          4A2-BRIP.sub.C277                                                                             187.0                                                         ______________________________________                                    

These unexpected results suggest that conjugates prepared with Type IRIP analogs according to the present invention may have enhancedstability and efficacy in vivo.

EXAMPLE 14

Plants of the genus Momordica produce a number of related proteins knownas momordins or momorcharins which are Type I RIPs. The gene encodingmomordin II was cloned from Momordica balsamina seeds.

Preparation of M. balsamina RNA

Total RNA was prepared from 4 g of M. balsamina seeds as described inAusubel et al., supra. PolyA containing RNA was prepared from 1 mg oftotal RNA by chromatography on oligo-(dT)-cellulose. 40 mg ofoligo-(dT)-cellulose Type 7 (Pharmacia) was added to 0.1 N NaOH andpoured into a disposable column (Biorad). The column was washed withwater until the eluate was pH 5.5, and then was washed with 1X loadingbuffer (50 mM NaCitrate, 0.5M NaCl, 1 mM EDTA, 0.1% SDS, pH 7.0) untilthe eluate was pH 7.0. 1 mg of total RNA was suspended in 300 μl ofwater, heated to 65° C. for 5 minutes, and 300 μl of 2X loading bufferwas added (100 mM Na Citrate, 1M NaCl, 2 mM EDTA, and 0.2% SDS). The RNAwas loaded onto the column, and the flow through was reheated to 65° C.,cooled to room temperature, and reloaded onto the column. Column-boundmRNA was washed 5 times with 0.5 ml of 1X loading buffer, and two timeswith 0.5 ml of 0.05M NaCitrate, 0.1 M NaCl, 1 mM EDTA, 0.1% SDS.PolyA-containing RNA was eluted two times from the column with 0.5 ml of25 mM NaCitrate, 1 mM EDTA, and 0.05% SDS.

Library Preparation

A cDNA library from the polyA-containing M. balsamina RNA was preparedin a bacterial expression plasmid with the SuperScript Plasmid System(BRL, Gaithersburg, Md.). The cDNA was synthesized from 2 μg of polyA-containing RNA, size fractionated, digested with NotI, and ligatedinto the expression vector pSPORT as recommended by the manufacturer ofthe vector, BRL.

Cloning of the Momordin II Gene

A DNA fragment encoding the first 27 amino acids of momordin II wasamplified from M. balsamina cDNA by PCR. First strand cDNA was preparedfrom 100 ng of polyA containing RNA with an RNA-PCR Kit (Perkin ElmerCetus). Two partially degenerate primers were synthesized based on theamino acid sequence of the first 27 amino acids of momordin II describedin Li et al., Experientia, 36, 524-527 (1980). Because the amino acidsequence of amino acids 1-27 of momordin II is 52% homologous to aminoacids 1-17 of momordin I [Ho et al., BBA, 1088, 311-314 (1991)], somecodon assignments in the degenerate primers were based on hornology tothe corresponding amino acid as well as codon preference in the momordinI gene. The sequences of primers momo-3 and momo-4 are set out belowusing IUPAC nucleotide symbols.

    ______________________________________                                        momo-3 (SEQ ID NO: 52)                                                        5' GATGTTAAYTTYGAYTTGTCNACDGCTAC 3'                                           momo-4 (SEQ ID NO: 53)                                                        5' ATTGGNAGDGTAGCCCTRAARTCYTCDAT 3'                                           ______________________________________                                    

The resulting 81 bp PCR product was purified on a 5% acrylamide gel andcloned into the Smal site of pUC18. Three candidate clones weresequenced, and one clone, pMO110, was identified which encoded theN-terminal 27 amino acids of momordin II.

A hybridization probe was designed for screening of the momordin II cDNAlibrary based on the sequence of the pMO110 momordin II DNA fragment.The sequence of the primer momo-5 is shown below.

    ______________________________________                                        momo-5 (SEQ ID NO: 54)                                                         ##STR29##                                                                    ______________________________________                                    

Primer momo-5 corresponds to amino acids 9-18 of mature momordin II. Theunderlined nucleotides of the primer were expected to match the DNAsequence of the momordin II gene exactly. Since this sequence is highlyA/T-rich and may hybridize to the momordin II gene weakly, theadditional adjacent nucleotides were included in the primer. Bases 3 and30 (overlined) were in the "wobble" position (i.e., the third nucleotidein a codon) of amino acids 9 (alanine) and 18 (isoleucine),respectively, of momordin II and may not be identical to the nucleotidebases in the native gene.

A 90,000 member cDNA library in pSPORT was screened with ³² P-kinasedmomo-5, and eight potential candidate clones were identified. One clone,pING3619, was sequenced and contains an open reading frame correspondingin part to the expected N-terminal 27 residues of Momordin II. Thecomplete momordin gene contains 286 amino acids, the first 23 of whichare a presumed leader signal (mature momordin II is 263 residues). TheDNA sequence of the momordin II gene is set out in SEQ ID NO: 13.

Construction of an Expression Vector Containing the Momordin II Gene

A bacterial expression vector for the momordin II gene was constructed.Two PCR primers were synthesized, one (momo-9) which primes from the +1residue of the mature momordin II amino acid sequence, and one at theC-terminus (momo-10) of momordin II which introduces an XhoI restrictionsite:

    ______________________________________                                        momo-9 (SEQ ID NO: 55)                                                        5' GATGTTAACTTCGATTTGTCGA 3'                                                  momo-10 (SEQ ID NO: 56)                                                       5' TCAACTCGAGGTACTCAATTCACAACAGATTCC 3'                                       ______________________________________                                         pING3619 was amplified with momo-9 and momo-10, and the product was     treated with T4 polymerase, cut with XhoI, and purified on an agarose gel.     This gene fragment was ligated along with the 131 bp pelB leader fragment     from pIC100 which has been generated by SstI digestion, T4-polymerase     treatment, and EcoRI digestion, into the arab expression vector cleaved     with EcoRI and XhoI. The product of this three piece ligation was     sequenced to verify that the pelB junction and momordin II coding sequence     were correct. Arabinose induction of cells containing the momordin II     expression plasmid pING3621 results in production of momordin II in E.     coli.

Analogs of Mormordin II

Mormordin II has no natural cysteines available for conjugation toantibody. Analogs of mormordin which have a free cysteine forconjugation to an antibody may be constructed. Positions likely to beappropriate for substitution of a cysteine residue may be identifiedfrom FIG. 3 as positions near the ricin A-chain cysteine₂₅₉ and aspositions including the last 26 amino acids of momordin II that areaccessible to solvent. For example, the arginine at position 242 ofmomordin II aligns with the ricin A-chain cysteine at position 259 andis a preferred target for substitution. Additional preferredsubstitution positions for momordin II include the serine at position241 and the alanine at position 243.

While the present invention has been described in terms of preferredembodiments, it is understood that variations and improvements willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations which come withinthe scope of the invention as claimed.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 101                                                (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 267 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       IlePheProLysGln TyrProIleIleAsnPheThrThrAlaGlyAla                             151015                                                                        ThrValGlnSerTyrThrAsnPheIleArgAlaValArgGlyArgLeu                              20 2530                                                                       ThrThrGlyAlaAspValArgHisGluIleProValLeuProAsnArg                              354045                                                                        ValGlyLeuProIleAsn GlnArgPheIleLeuValGluLeuSerAsn                             505560                                                                        HisAlaGluLeuSerValThrLeuAlaLeuAspValThrAsnAlaTyr                              6570 7580                                                                     ValValGlyTyrArgAlaGlyAsnSerAlaTyrPhePheHisProAsp                              859095                                                                        AsnGlnGluAspAlaGlu AlaIleThrHisLeuPheThrAspValGln                             100105110                                                                     AsnArgTyrThrPheAlaPheGlyGlyAsnTyrAspArgLeuGluGln                              115 120125                                                                    LeuAlaGlyAsnLeuArgGluAsnIleGluLeuGlyAsnGlyProLeu                              130135140                                                                     GluGluAlaIleSerAlaLeuTyrTyrT yrSerThrGlyGlyThrGln                             145150155160                                                                  LeuProThrLeuAlaArgSerPheIleIleCysIleGlnMetIleSer                              165 170175                                                                    GluAlaAlaArgPheGlnTyrIleGluGlyGluMetArgThrArgIle                              180185190                                                                     ArgTyrAsnArgArgSerAl aProAspProSerValIleThrLeuGlu                             195200205                                                                     AsnSerTrpGlyArgLeuSerThrAlaIleGlnGluSerAsnGlnGly                              210215 220                                                                    AlaPheAlaSerProIleGlnLeuGlnArgArgAsnGlySerLysPhe                              225230235240                                                                  SerValTyrAspValSerIleLeu IleProIleIleAlaLeuMetVal                             245250255                                                                     TyrArgCysAlaProProProSerSerGlnPhe                                             260265                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 251 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GlyLeuAspThrValSerPheSerThrLysGlyAlaThrTyrIleThr                              15 1015                                                                       TyrValAsnPheLeuAsnGluLeuArgValLysLeuLysProGluGly                              202530                                                                        AsnSerHisGlyIlePro LeuLeuArgLysLysCysAspAspProGly                             354045                                                                        LysCysPheValLeuValAlaLeuSerAsnAspAsnGlyGlnLeuAla                              5055 60                                                                       GluIleAlaIleAspValThrSerValTyrValValGlyTyrGlnVal                              65707580                                                                      ArgAsnArgSerTyrPhePheLys AspAlaProAspAlaAlaTyrGlu                             859095                                                                        GlyLeuPheLysAsnThrIleLysThrArgLeuHisPheGlyGlyThr                              100 105110                                                                    TyrProSerLeuGluGlyGluLysAlaTyrArgGluThrThrAspLeu                              115120125                                                                     GlyIleGluProLeuArgIleGlyIl eLysLysLeuAspGluAsnAla                             130135140                                                                     IleAspAsnTyrLysProThrGluIleAlaSerSerLeuLeuValVal                              145150 155160                                                                 IleGlnMetValSerGluAlaAlaArgPheThrPheIleGluAsnGln                              165170175                                                                     IleArgAsnAsnPheGlnGlnArg IleArgProAlaAsnAsnThrIle                             180185190                                                                     SerLeuGluAsnLysTrpGlyLysLeuSerPheGlnIleArgThrSer                              195 200205                                                                    GlyAlaAsnGlyMetPheSerGluAlaValGluLeuGluArgAlaAsn                              210215220                                                                     GlyLysLysTyrTyrValThrAlaValAspGln ValLysProLysIle                             225230235240                                                                  AlaLeuLeuLysPheValAspLysAspProLys                                             245250                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 280 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AlaAlaLysMetAlaLysAsnValAspLysProLeuPheThrAlaThr                              15 1015                                                                       PheAsnValGlnAlaSerSerAlaAspTyrAlaThrPheIleAlaGly                              202530                                                                        IleArgAsnLysLeuArgAsnP roAlaHisPheSerHisAsnArgPro                             354045                                                                        ValLeuProProValGluProAsnValProProSerArgTrpPheHis                              5055 60                                                                       ValValLeuLysAlaSerProThrSerAlaGlyLeuThrLeuAlaIle                              65707580                                                                      ArgAlaAspAsnIleTyrLeuGluGlyP heLysSerSerAspGlyThr                             859095                                                                        TrpTrpGluLeuThrProGlyLeuIleProGlyAlaThrTyrValGly                              100 105110                                                                    PheGlyGlyThrTyrArgAspLeuLeuGlyAspThrAspLysLeuThr                              115120125                                                                     AsnValAlaLeuGlyArgGlnGlnLeuAla AspAlaValThrAlaLeu                             130135140                                                                     HisGlyArgThrLysAlaAspLysAlaSerGlyProLysGlnGlnGln                              145150155 160                                                                 AlaArgGluAlaValThrThrLeuValLeuMetValAsnGluAlaThr                              165170175                                                                     ArgPheGlnThrValSerGlyPheVal AlaGlyLeuLeuHisProLys                             180185190                                                                     AlaValGluLysLysSerGlyLysIleGlyAsnGluMetLysAlaGln                              195200 205                                                                    ValAsnGlyTrpGlnAspLeuSerAlaAlaLeuLeuLysThrAspVal                              210215220                                                                     LysProProProGlyLysSerProAlaLysPheAlaP roIleGluLys                             225230235240                                                                  MetGlyValArgThrAlaGluGlnAlaAlaAsnThrLeuGlyIleLeu                              24525 0255                                                                    LeuPheValGluValProGlyGlyLeuThrValAlaLysAlaLeuGlu                              260265270                                                                     LeuPheHisAlaSerGlyGlyLys                                                       275280                                                                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 263 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AspValAsnPheAspLeuSerThrAlaThrAlaLysThr TyrThrLys                             151015                                                                        PheIleGluAspPheArgAlaThrLeuProPheSerHisLysValTyr                              2025 30                                                                       AspIleProLeuLeuTyrSerThrIleSerAspSerArgArgPheIle                              354045                                                                        LeuLeuAspLeuThrSerTyrAlaTyrGluThrIleSerVal AlaIle                             505560                                                                        AspValThrAsnValTyrValValAlaTyrArgThrArgAspValSer                              65707580                                                                      TyrPhePheLysGluSerProProGluAlaTyrAsnIleLeuPheLys                              859095                                                                        GlyThrArgLysIleThrLeuProTyrThrGlyAsnTyrGlu AsnLeu                             100105110                                                                     GlnThrAlaAlaHisLysIleArgGluAsnIleAspLeuGlyLeuPro                              115120125                                                                      AlaLeuSerSerAlaIleThrThrLeuPheTyrTyrAsnAlaGlnSer                             130135140                                                                     AlaProSerAlaLeuLeuValLeuIleGlnThrThrAlaGluAlaAla                               145150155160                                                                 ArgPheLysTyrIleGluArgHisValAlaLysTyrValAlaThrAsn                              16517017 5                                                                    PheLysProAsnLeuAlaIleIleSerLeuGluAsnGlnTrpSerAla                              180185190                                                                     LeuSerLysGlnIlePheLeuAlaGlnAsnGlnGlyGlyLysPh eArg                             195200205                                                                     AsnProValAspLeuIleLysProThrGlyGluArgPheGlnValThr                              210215220                                                                     Asn ValAspSerAspValValLysGlyAsnIleLysLeuLeuLeuAsn                             225230235240                                                                  SerArgAlaSerThrAlaAspGluAsnPheIleThrThrMetThrLeu                              245250255                                                                     LeuGlyGluSerValValAsn                                                         260                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 248 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       AspValArgPheSerLeuSerGlySerSerSerThrSerTyrSerLys                              151015                                                                        PheIleGlyAspLeuArg LysAlaLeuProSerAsnGlyThrValTyr                             202530                                                                        AsnLeuThrIleLeuLeuSerSerAlaSerGlyAlaSerArgTyrThr                              35 4045                                                                       LeuMetThrLeuSerAsnTyrAspGlyLysAlaIleThrValAlaVal                              505560                                                                        AspValSerGlnLeuTyrIleMetGlyTyr LeuValAsnSerThrSer                             65707580                                                                      TyrPhePheAsnGluSerAspAlaLysLeuAlaSerGlnTyrValPhe                              85 9095                                                                       LysGlySerThrIleValThrLeuProTyrSerGlyAsnTyrGluLys                              100105110                                                                     LeuGlnThrAlaAlaGlyLysIle ArgGluLysIleProLeuGlyPhe                             115120125                                                                     ProAlaLeuAspSerAlaLeuThrThrIlePheHisTyrAspSerThr                              130135 140                                                                    AlaAlaAlaAlaAlaPheLeuValIleLeuGlnThrThrAlaGluAla                              145150155160                                                                  SerArgPheLysTyrIleGluGlyGln IleIleGluArgIleSerLys                             165170175                                                                     AsnGlnValProSerLeuAlaThrIleSerLeuGluAsnSerLeuTrp                              180 185190                                                                    SerAlaLeuSerLysGlnIleGlnLeuAlaGlnThrAsnAsnGlyThr                              195200205                                                                     PheLysThrProValValIleThrAspA spLysGlyGlnArgValGlu                             210215220                                                                     IleThrAsnValThrSerLysValValThrLysAsnIleGlnLeuLeu                              22523023 5240                                                                 LeuAsnTyrLysGlnAsnValAla                                                      245                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 255 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        AspValSerPheArgLeuSerGlyAlaThrSerSerSerTyrGlyVal                             151015                                                                        PheIleSerAsnLeuArgLysAlaLeuProAsnGluArgLysLeuT yr                             202530                                                                        AspLeuProLeuIleArgSerSerLeuProGlySerGlnArgTyrAla                              354045                                                                        I leIleHisLeuThrAsnTyrAlaAspGluValAlaLeuAspValThr                             505560                                                                        AsnValAspAlaGlyLeuProArgAsnAlaValLeuTyrIleMetGly                              65 707580                                                                     TyrArgAlaGlyAspThrSerTyrPhePheAsnGluAlaSerAlaThr                              859095                                                                        G luAlaAlaLysTyrValPheLysAspAlaMetArgLysValThrLeu                             100105110                                                                     ProTyrSerGlyAsnTyrGluArgLeuGlnThrAlaAlaGlyGlyLeu                               115120125                                                                    ArgGluAsnIleProLeuGlyLeuProAlaLeuAspSerAlaIleThr                              130135140                                                                     ThrLeuPheTy rTyrAsnAlaAsnSerAlaAlaSerAlaLeuMetVal                             145150155160                                                                  LeuIleGlnSerThrSerGluAlaAlaArgTyrLysPheIleGluGln                               165170175                                                                    GlnIleGlySerArgValAspLysThrPheLeuProSerLeuAlaIle                              180185190                                                                     Ile SerLeuGluAsnSerLeuTrpLeuAlaLeuSerLysGlnIleGln                             195200205                                                                     IleAlaSerThrAsnAsnGlyGluPheGluThrProValValLeuIle                               210215220                                                                    AsnAlaGlnAsnGlnArgValThrIleThrAsnValAspAlaGlyVal                              225230235240                                                                  ValThr SerAsnIleAlaLeuLeuLeuAsnArgAsnAsnMetAla                                245250255                                                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 263 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AspValSerPheArgLeuSerGlyAlaAspProArgSerTyrGlyMet                              151015                                                                        PheIleLysAspLeuArgAsnAl aLeuProPheArgGluLysValTyr                             202530                                                                        AsnIleProLeuLeuLeuProSerValSerGlyAlaGlyArgTyrLeu                              35 4045                                                                       LeuMetHisLeuPheAsnTyrAspGlyLysThrIleThrValAlaVal                              505560                                                                        AspValThrAsnValTyrIleMetGlyTyrLeuAl aAspThrThrSer                             65707580                                                                      TyrPhePheAsnGluProAlaAlaGluLeuAlaSerGlnTyrValPhe                              85 9095                                                                       ArgAspAlaArgArgLysIleThrLeuProTyrSerGlyAsnTyrGlu                              100105110                                                                     ArgLeuGlnIleAlaAlaGlyLysProA rgGluLysIleProIleGly                             115120125                                                                     LeuProAlaLeuAspSerAlaIleSerThrLeuLeuHisTyrAspSer                              130135 140                                                                    ThrAlaAlaAlaGlyAlaLeuLeuValLeuIleGlnThrThrAlaGlu                              145150155160                                                                  AlaAlaArgPheLysTyrIleGluGlnGlnIl eGlnGluArgAlaTyr                             165170175                                                                     ArgAspGluValProSerLeuAlaThrIleSerLeuGluAsnSerTrp                              180185 190                                                                    SerGlyLeuSerLysGlnIleGlnLeuAlaGlnGlyAsnAsnGlyIle                              195200205                                                                     PheArgThrProIleValLeuValAspAsnLys GlyAsnArgValGln                             210215220                                                                     IleThrAsnValThrSerLysValValThrSerAsnIleGlnLeuLeu                              225230235 240                                                                 LeuAsnThrArgAsnIleAlaGluGlyAspAsnGlyAspValSerThr                              245250255                                                                     ThrHisGlyPheSerSerThr                                                          260                                                                          (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 250 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AlaProThrLeuGluThrIleAlaSerLeuAspLeuAsnAsnProThr                              1 51015                                                                       ThrTyrLeuSerPheIleThrAsnIleArgThrLysValAlaAspLys                              202530                                                                        ThrGl uGlnCysThrIleGlnLysIleSerLysThrPheThrGlnArg                             354045                                                                        TyrSerTyrIleAspLeuIleValSerSerThrGlnLysIleThrLeu                              50 5560                                                                       AlaIleAspMetAlaAspLeuTyrValLeuGlyTyrSerAspIleAla                              65707580                                                                      AsnAsnLysGl yArgAlaPhePhePheLysAspValThrGluAlaVal                             859095                                                                        AlaAsnAsnPhePheProGlyAlaThrGlyThrAsnArgIleLysLeu                               100105110                                                                    ThrPheThrGlySerTyrGlyAspLeuGluLysAsnGlyGlyLeuArg                              115120125                                                                     LysAspAsnPro LeuGlyIlePheArgLeuGluAsnSerIleValAsn                             130135140                                                                     IleTyrGlyLysAlaGlyAspValLysLysGlnAlaLysPhePheLeu                              1451 50155160                                                                 LeuAlaIleGlnMetValSerGluAlaAlaArgPheLysTyrIleSer                              165170175                                                                     AspLysIleP roSerGluLysTyrGluGluValThrValAspGluTyr                             180185190                                                                     MetThrAlaLeuGluAsnAsnTrpAlaLysLeuSerThrAlaValTyr                              19 5200205                                                                    AsnSerLysProSerThrThrThrAlaThrLysCysGlnLeuAlaThr                              210215220                                                                     SerProValThrIleSerPr oTrpIlePheLysThrValGluGluIle                             225230235240                                                                  LysLeuValMetGlyLeuLeuLysSerSer                                                245250                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 261 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       IleAsnThrIleThrPheAspAlaGlyAsnAlaThrIleAsnLysTyr                              1 51015                                                                       AlaThrPheMetGluSerLeuArgAsnGluAlaLysAspProSerLeu                              202530                                                                        LysCysTyrGly IleProMetLeuProAsnThrAsnSerThrIleLys                             354045                                                                        TyrLeuLeuValLysLeuGlnGlyAlaSerLeuLysThrIleThrLeu                              50 5560                                                                       MetLeuArgArgAsnAsnLeuTyrValMetGlyTyrSerAspProTyr                              65707580                                                                      AspAsnLysCysArgTyr HisIlePheAsnAspIleLysGlyThrGlu                             859095                                                                        TyrSerAspValGluAsnThrLeuCysProSerSerAsnProArgVal                              100 105110                                                                    AlaLysProIleAsnTyrAsnGlyLeuTyrProThrLeuGluLysLys                              115120125                                                                     AlaGlyValThrSerArgA snGluValGlnLeuGlyIleGlnIleLeu                             130135140                                                                     SerSerLysIleGlyLysIleSerGlyGlnGlySerPheThrGluLys                              145150 155160                                                                 IleGluAlaAspPheLeuLeuValAlaIleGlnMetValSerGluAla                              165170175                                                                     AlaArgPheLysTyrIl eGluAsnGlnValLysThrAsnPheAsnArg                             180185190                                                                     AspPheSerProAsnAspLysValLeuAspLeuGluGluAsnTrpGly                              195 200205                                                                    LysIleSerThrAlaIleHisAsnSerLysAsnGlyAlaLeuProLys                              210215220                                                                     ProLeuGluLeuLysAsnAlaAspGly ThrLysTrpIleValLeuArg                             225230235240                                                                  ValAspGluIleLysProAspValGlyLeuLeuAsnTyrValAsnGly                              245 250255                                                                    ThrCysGlnAlaThr                                                               260                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 259 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       ValThrSerIleThrLeuAspLeuValAsnProThrAlaGlyGlnTyr                             151015                                                                        SerSerPheValAspLysIleArgAsnAsnValLysAspProAsnLeu                              202530                                                                        LysTyrGlyGlyThrAspIleAlaValIleGlyProProSerLysGlu                              354045                                                                        Lys PheLeuArgIleAsnPheGlnSerSerArgGlyThrValSerLeu                             505560                                                                        GlyLeuLysArgAspAsnLeuTyrValValAlaTyrLeuAlaMetAsp                              65 707580                                                                     AsnThrAsnValAsnArgAlaTyrTyrPheArgSerGluIleThrSer                              859095                                                                        Ala GluSerThrAlaLeuPheProGluAlaThrThrAlaAsnGlnLys                             100105110                                                                     AlaLeuGluTyrThrGluAspTyrGlnSerIleGluLysAsnAlaGln                               115120125                                                                    IleThrGlnGlyAspGlnSerArgLysGluLeuGlyLeuGlyIleAsp                              130135140                                                                     LeuLeuSerThr SerMetGluAlaValAsnLysLysAlaArgValVal                             145150155160                                                                  LysAspGluAlaArgPheLeuLeuIleAlaIleGlnMetThrAlaGlu                               165170175                                                                    AlaAlaArgPheArgTyrIleGlnAsnLeuValIleLysAsnPhePro                              180185190                                                                     AsnL ysPheAsnSerGluAsnLysValIleGlnPheGluValAsnTrp                             195200205                                                                     LysLysIleSerThrAlaIleTyrGlyAspAlaLysAsnGlyValPhe                              21 0215220                                                                    AsnLysAspTyrAspPheGlyPheGlyLysValArgGlnValLysAsp                              225230235240                                                                  LeuGlnMe tGlyLeuLeuMetTyrLeuGlyLysProLysSerSerAsn                             245250255                                                                     GluAlaAsn                                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 813 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GGGCTAGATACCGTGTCATTCTCAACCAAAGGTGCCACTTATATTACCTACGTGAATTTC60                TTGAATGAGCTACGAGTTAAATTGAAACCCGAAGGTAACAGCCATGGAATCCC ATTGCTG120              CGCAAAAAATGTGATGATCCTGGAAAGTGTTTCGTTTTGGTAGCGCTTTCAAATGACAAT180               GGACAGTTGGCGGAAATAGCTATAGATGTTACAAGTGTTTATGTGGTGGGCTATCAAGTA240               AGAAACAGATCTTACTTCTTTAAAGATGCT CCAGATGCTGCTTACGAAGGCCTCTTCAAA300              AACACAATTAAAACAAGACTTCATTTTGGCGGCACGTATCCCTCGCTGGAAGGTGAGAAG360               GCATATAGAGAGACAACAGACTTGGGCATTGAACCATTAAGGATTGGCATCAAGAAACTT420               GATGAAAA TGCGATAGACAATTATAAACCAACGGAGATAGCTAGTTCTCTATTGGTTGTT480              ATTCAAATGGTGTCTGAAGCAGCTCGATTCACCTTTATTGAGAACCAAATTAGAAATAAC540               TTTCAACAGAGAATTCGCCCGGCGAATAATACAATCAGCCTTGAGAATAAATG GGGTAAA600              CTCTCGTTCCAGATCCGGACATCAGGTGCAAATGGAATGTTTTCGGAGGCAGTTGAATTG660               GAACGTGCAAATGGCAAAAAATACTATGTCACCGCAGTTGATCAAGTAAAACCCAAAATA720               GCACTCTTGAAGTTCGTCGATAAAGATCCT AAAACGAGCCTTGCTGCTGAATTGATAATC780              CAGAACTATGAGTCATTAGTGGGCTTTGATTAG813                                          (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 846 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ATGGCGGCAAAGATGGCGAAGAACGTGGACAAGCCGCTCTTCACCGCGACGTTCAACGTC60                CAGGCCAGCTCCGCCGACTACGCCACCTTCATCGCCGGCATCCGCAACAAGCTCCGCAAC120               CCGGCGC ACTTCTCCCACAACCGCCCCGTGCTGCCGCCGGTCGAGCCCAACGTCCCGCCG180              AGCAGGTGGTTCCACGTCGTGCTCAAGGCCTCGCCGACCAGCGCCGGGCTCACGCTGGCC240               ATCCGCGCGGACAACATCTACCTGGAGGGCTTCAAGAGCAGCGACGGCACCT GGTGGGAG300              CTCACCCCGGGCCTCATCCCCGGCGCCACCTACGTCGGGTTCGGCGGCACCTACCGCGAC360               CTCCTCGGCGACACCGACAAGCTAACCAACGTCGCTCTCGGCCGACAGCAGCTGGCGGAC420               GCGGTGACCGCGCTCCACGGGCGCACCAAG GCCGACAAGGCCTCCGGCCCGAAGCAGCAG480              CAGGCGAGGGAGGCGGTGACGACGCTGGTCCTCATGGTGAACGAGGCCACGCGGTTCCAG540               ACGGTGTCTGGGTTCGTGGCCGGGTTGCTGCACCCCAAGGCGGTGGAGAAGAAGAGCGGG600               AAGATCG GCAATGAGATGAAGGCCCAGGTGAACGGGTGGCAGGACCTGTCCGCGGCGCTG660              CTGAAGACGGACGTGAAGCCTCCGCCGGGAAAGTCGCCAGCGAAGTTCGCGCCGATCGAG720               AAGATGGGCGTGAGGACGGCTGAACAGGCCGCCAACACGCTGGGGATCCTGC TGTTCGTG780              GAGGTGCCGGGTGGGTTGACGGTGGCCAAGGCGCTGGAGCTGTTCCATGCGAGTGGTGGG840               AAATAG846                                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 913 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CGTCCGAAAATGGTGAAATGCTTACTACTTTCTTTTTTAATTATCGCCATCTTCATTGGT60                GTTCCTACTGCCAAAGGCGATGTTAACTT CGATTTGTCGACTGCCACTGCAAAAACCTAC120              ACAAAATTTATCGAAGATTTCAGGGCGACTCTTCCATTTAGCCATAAAGTGTATGATATA180               CCTCTACTGTATTCCACTATTTCCGACTCCAGACGTTTCATACTCCTCGATCTTACAAGT240               TATGCA TATGAAACCATCTCGGTGGCCATAGATGTGACGAACGTTTATGTTGTGGCGTAT300              CGCACCCGCGATGTATCCTACTTTTTTAAAGAATCTCCTCCTGAAGCTTATAACATCCTA360               TTCAAAGGTACGCGGAAAATTACACTGCCATATACCGGTAATTATGAAAAT CTTCAAACT420              GCTGCACACAAAATAAGAGAGAATATTGATCTTGGACTCCCTGCCTTGAGTAGTGCCATT480               ACCACATTGTTTTATTACAATGCCCAATCTGCTCCTTCTGCATTGCTTGTACTAATCCAG540               ACGACTGCAGAAGCTGCAAGATTTAAGTA TATCGAGCGACACGTTGCTAAGTATGTTGCC600              ACTAACTTTAAGCCAAATCTAGCCATCATAAGCTTGGAAAATCAATGGTCTGCTCTCTCC660               AACAAATCTTTTTGGCGCAGAATCAAGGAGGAAAATTTAGAAATCCTGTCGACCTTATAA720               AACCTA CCGGGGAACGGTTTCAAGTAACCAATGTTGATTCAGATGTTGTAAAAGGTAATA780              TCAAACTCCTGCTGAACTCCAGAGCTAGCACTGCTGATGAAAACTTTATCACAACCATGA840               CTCTACTTGGGGAATCTGTTGTGAATTGAAAGTTTAATAATCCACCCATAT CGAAATAAG900              GCATGTTCATGAC913                                                              (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      TTYAARGAYGCNCCNGAYGCNGCNTAYGARGG32                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ACYTGRTCNACNGCNGTNACRTARTAYTTYTT32                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GGNYTNGAYACNGTNWSNTTYWSNACNAARGG32                                            (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii ) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      AATGGTTCAATGCCCAAGTCTGT23                                                     (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (i i) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      TGTCTCTCTATATGCCTTTCCAC23                                                     (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          ( ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      TCAACCCGGGCTAGATACCGTGTCATTCTCAACCAAAGGTGCCACTTATATTA53                       (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CTTCATTTTGGCGGCACGTATCC23                                                     (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CTCGAGGCTGCAAGCTTACGTGGGATTTTTTTTTTTTTTTTTTTTT46                              (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CTCGCTGGAAGGTGAGAA18                                                          (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CTCGAGGCTGCAAGCTTACGTGGGA25                                                   (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      TGATCTCGAGTACTATTTAGGATCTTTATCGACGA35                                         (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      GTAAGCAGCATCTGGAGCATCT22                                                      (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CATTCAAGAAATTCACGTAGG21                                                       (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      GGCCTGGACACCGTGAGCTTTAG23                                                     (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      TCGATTGCGATCCTAAATAGTACTC25                                                   (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TTTAGGATCGCAATCGACGAACTTCAAG28                                                (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D ) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      GTTCGTCTGTAAAGATCCTAAATAGTACTCGA32                                            (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      ( D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GGATCTTTACAGACGAACTTCAAGAGT27                                                 (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      TCTTGTGCTTCGTCGATAAAGATCC25                                                   (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      ATCGACGAAGCACAAGAGTGCTATTTT27                                                 (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      GTAAAACCATGCATAGCACTCTTGAAGTTCGT32                                            (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      AGTGCTATGCATGGTTTTACTTGATCAACTGC32                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      AGCACATGTGGTGCCACTTATATTACCTA29                                               (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      TAAGTGGCACCACATGTGCTAAAGCTCACGGTG33                                           (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      TGACTGTGGACAGTTGGCGGAAATA25                                                   (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      GCCACTGTCCACAGTCATTTGAAAGCGCTACC32                                            (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      GATGATCCTGGAAAGGCTTTCGTTTTGGTAGCGCTT36                                        (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      AAGCCTTTCCAGGATCATCAGCTTTTTTGGGCAGCAATGGG41                                   (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      AAGCCTTTCCAGGATCATCACAT23                                                     (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      GCGACTCTCTACTGTTTC18                                                          (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      CGTTAGCAATTTAACTGTGAT21                                                       (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      AACAGCTATGACCATG16                                                            (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      TGAACTCGAGGAAACTACCTATTTCCCAC29                                               (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C ) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      GCATTACATCCATGGCGGC19                                                         (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 64 base pairs                                                     (B) TYPE: nucleic acid                                                        ( C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      GATATCTCGAGTTAACTATTTCCCACCACACGCATGGAACAGCTCCAGCGCCTTGGCCAC60                CGTC6 4                                                                       (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      TATCTGTTCGTGGAGGTGCCG 21                                                      (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      CGTTAGCAATTTAACTGTGAT 21                                                      (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      CCAAGTGTCTGGAGCTGTTCCATGCGA 27                                                (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      GATGTTAAYTTYGAYTTGTCNACDGCTAC 29                                              (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      ATTGGNAGDGTAGCCCTRAARTCYTCDAT 29                                              (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      GCCACTGCAAAAACCTACACAAAATTTATTGA 32                                           (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      GATGTTAACTTCGATTTGTCGA 22                                                     (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      TCAACTCGAGGTACTCAATTCACAACAGATTCC 33                                          (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 813 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      GGCCTGGACACCGTGAGCTTTAGCACTAAAGGTGCCACTTATATTACCTACGTGAAT TTC60               TTGAATGAGCTACGAGTTAAATTGAAACCCGAAGGTAACAGCCATGGAATCCCATTGCTG120               CGCAAAAAATGTGATGATCCTGGAAAGTGTTTCGTTTTGGTAGCGCTTTCAAATGACAAT180               GGACAGTTGGCGGAAATAGCTATAGATGTTACAA GTGTTTATGTGGTGGGCTATCAAGTA240              AGAAACAGATCTTACTTCTTTAAAGATGCTCCAGATGCTGCTTACGAAGGCCTCTTCAAA300               AACACAATTAAAACAAGACTTCATTTTGGCGGCACGTATCCCTCGCTGGAAGGTGAGAAG360               GCATATAGAGA GACAACAGACTTGGGCATTGAACCATTAAGGATTGGCATCAAGAAACTT420              GATGAAAATGCGATAGACAATTATAAACCAACGGAGATAGCTAGTTCTCTATTGGTTGTT480               ATTCAAATGGTGTCTGAAGCAGCTCGATTCACCTTTATTGAGAACCAAATTAGAAAT AAC540              TTTCAACAGAGAATTCGCCCGGCGAATAATACAATCAGCCTTGAGAATAAATGGGGTAAA600               CTCTCGTTCCAGATCCGGACATCAGGTGCAAATGGAATGTTTTCGGAGGCAGTTGAATTG660               GAACGTGCAAATGGCAAAAAATACTATGTCACCG CAGTTGATCAAGTAAAACCCAAAATA720              GCACTCTTGAAGTTCGTCGATAAAGATCCTAAAACGAGCCTTGCTGCTGAATTGATAATC780               CAGAACTATGAGTCATTAGTGGGCTTTGATTAG813                                          (2) INFORMATION FOR SEQ ID NO:58:                                              (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      CysHisHisHisAlaSerArgValAlaArgMetAlaSerAspGluPhe                              15 1015                                                                       ProSerMetCys                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      ProSerG lyGlnAlaGlyAlaAlaAlaSerGluSerLeuPheIleSer                             151015                                                                        AsnHisAlaTyr                                                                  20                                                                            (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      CAGCCATGGAATCCCATTGCTG22                                                      (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 28 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      CACATGTAAAACAAGACTTCATTTTGGC28                                                (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 36 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      TGAAGTCTTGTTTTAGATGTGTTTTTGAAGAGGCCT36                                        (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      ATGCCATATGCAATTATAAACCAACGGAGA30                                              (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 39 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      GGTTTATAATTGCATATGGCATTTTCATCAAGTTTCTTG39                                     (2) INFORMATION FOR SEQ ID NO:65:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                      CTTTCAACAATGCATTCGCCCGGCGAATAATAC33                                           (2) INFORMATION FOR SEQ ID NO:66:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 33 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                      GCGAATGCATTGTTGAAAGTTATTTCTAATTTG33                                           (2) INFORMATION FOR SEQ ID NO:67:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                      GTTTTGTGAGGCAGTTGAATTGGAAC26                                                  (2) INFORMATION FOR SEQ ID NO:68:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                      TTCAACTGCCTCACAAAACATTCCATTTGCACCT34                                          (2) INFORMATION FOR SEQ ID NO:69:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 24 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                      AAAAGCTGATGATCCTGGAAAGTG24                                                    (2) INFORMATION FOR SEQ ID NO:70:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 35 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                      TCCAGGATCATCAGCTTTTTTGCGCAGCAATGGGA35                                         (2) INFORMATION FOR SEQ ID NO:71:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 321 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                      GACATCCAGATGACTCAGTCTCCATCTTCCATGTCTGCATCTCTGGGAGACAGAGTCACT60                ATCACTTGCCGGGCGAGTCA GGACATTAATAGCTATTTAAGCTGGTTCCAGCAGAAACCA120              GGGAAATCTCCTAAGACCCTGATCTATCGTGCAAACAGATTGGTAGATGGGGTCCCATCA180               AGGTTCAGTGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGCAGCCTGCAATAT240               GAAGATTTTGGAATTTATTATTGTCAACAGTATGATGAGTCTCCGTGGACGTTCGGTGGA300               GGCACCAAGCTTGAAATCAAA321                                                      (2) INFORMATION FOR SEQ ID NO:72:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 354 base pairs                                                     (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                      CAGATCCAGTTGGTGCAGTCTGGACCTGGCCTGAAGAAGCCTGGAGGGTCCGTCAGAATC60                TCCTGCGCAGCTTCTGGGTATACCTTCACAAACTATGGAATG AACTGGGTGAAGCAGGCT120              CCAGGAAAGGGTTTAAGGTGGATGGGCTGGATAAACACCCACACTGGAGAGCCAACATAT180               GCTGATGACTTCAAGGGACGGTTTACCTTCTCTTTGGACACGTCTAAGAGCACTGCCTAT240               TTACAGATCAACAGCCTCAG AGCCGAGGACACGGCTACATATTTCTGTACAAGACGGGGT300              TACGACTGGTACTTCGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCC354                     (2) INFORMATION FOR SEQ ID NO:73:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 354 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                      GAGATCCAGTTGGTGCAGTCTGGAGGAGGCCTGGTGAAGCCTGGAGGGTCCGTCAGAATC60                TCCTGCGCAGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGGTGCGCCAGGCT1 20              CCAGGAAAGGGTTTAGAGTGGATGGGCTGGATAAACACCCACACTGGAGAGCCAACATAT180               GCTGATTCTTTCAAGGGACGGTTTACCTTCTCTTTGGACGATTCTAAGAACACTGCCTAT240               TTACAGATCAACAGCCTCAGAGCCGAGGACACGGCTGTGTA TTTCTGTACAAGACGGGGT300              TACGACTGGTACTTCGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCC354                     (2) INFORMATION FOR SEQ ID NO:74:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 321 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: DNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                      GACATCCAGATGACTCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGTCACT60                ATCACTTGCCGGGCGAGTCAGGACATTAATAGCTATTTAAGCTGGTTCCAGCAGAAACCA120               GGGAAAGCTCCTAAGACC CTGATCTATCGTGCAAACAGATTGGAATCTGGGGTCCCATCA180              AGGTTCAGTGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGCAGCCTGCAATAT240               GAAGATTTTGGAATTTATTATTGTCAACAGTATGATGAGTCTCCGTGGACGTTCGGTGGA 300              GGCACCAAGCTTGAAATCAAA321                                                      (2) INFORMATION FOR SEQ ID NO:75:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                      TGTCATCATCATGCATCGCGAGTTGCCAGAATGGCATCTGATGAGTTTCCTTCTATGTGC60                GCAAGTACTC70                                                                  (2) INFORMATION FOR SEQ ID NO:76:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                      TCGAGAGTACTTGCGCACATAGAAGGAAACTCATCAGATGCCATTCTGGCAACTCGCGAT60                GCATGATGATGACATGCA 78                                                         (2) INFORMATION FOR SEQ ID NO:77:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                      TGTTCGGCCGCATGTCATCATCATGCATCG 30                                             (2) INFORMATION FOR SEQ ID NO:78:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                      AGTCATGCCCCGCGC 15                                                            (2) INFORMATION FOR SEQ ID NO:79:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                      TCCCGGCTGTCCTACAGT 18                                                         (2) INFORMATION FOR SEQ ID NO:80:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                      TCCAGCCTGTCCAGATGGTGTGTGAGTTTTGTCAC AA37                                      (2) INFORMATION FOR SEQ ID NO:81:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 76 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                      CTAACTCGAGAGTACTGTATGCATGGTTCGAGAT GAACAAAGATTCTGAGGCTGCAGCTC60               CAGCCTGTCCAGATGG76                                                            (2) INFORMATION FOR SEQ ID NO:82:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                      CTAACTCGAGAGTACTGTAT20                                                        (2) INFORMATION FOR SEQ ID NO:83:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                      TCCAGCCTGTCCAGATGGACACTCTCCCCTGTTGAA36                                        (2) INFORMATION FOR SEQ ID NO:84:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                      GTACAGTGGAAGGTGGAT18                                                          (2) INFORMATION FOR SEQ ID NO:85:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                      CATGCGGCCGATTTAGGATCTTTATCGACGA31                                             (2) INFORMATION FOR SEQ ID NO:86:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                      AACATCCAGTTGGTGCAGTCTG22                                                      (2) INFORMATION FOR SEQ ID NO:87:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                      GAGGAGACGGTGACCGTGGT20                                                        (2) INFORMATION FOR SEQ ID NO:88:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                      GACATCAAGATGACCCAGT19                                                         (2) INFORMATION FOR SEQ ID NO:89:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                      GTTTGATTTCAAGCTTGGTGC21                                                       (2) INFORMATION FOR SEQ ID NO:90:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                      ACTTCGGCCGCACCATCTGGACAGGCTGGAG31                                             (2) INFORMATION FOR SEQ ID NO:91:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                      CGTTAGCAATTTAACTGTGAT21                                                       (2) INFORMATION FOR SEQ ID NO:92:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 723 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                      GACATCCAGATGACTCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGTCACT60                ATCACTTGCCGGGCGAGTCAGGACATTAATAGCTATTTAAGCTGGTTCCAGCAGAAACCA120               G GGAAAGCTCCTAAGACCCTGATCTATCGTGCAAACAGATTGGAATCTGGGGTCCCATCA180              AGGTTCAGTGGCAGTGGATCTGGGACAGATTATACTCTCACCATCAGCAGCCTGCAATAT240               GAAGATTTTGGAATTTATTATTGTCAACAGTATGATGAGTCTCCGTG GACGTTCGGTGGA300              GGCACCAAGCTTGAGATGAAAGGTGGCGGTGGATCTGGTGGAGGTGGGTCCGGAGGTGGA360               GGATCTGAGATCCAGTTGGTGCAGTCTGGAGGAGGCCTGGTGAAGCCTGGAGGGTCCGTC420               AGAATCTCCTGCGCAGCTTCTGGG TATACCTTCACAAACTATGGAATGAACTGGGTGCGC480              CAGGCTCCAGGAAAGGGTTTAGAGTGGATGGGCTGGATAAACACCCACACTGGAGAGCCA540               ACATATGCTGATTCTTTCAAGGGACGGTTTACCTTCTCTTTGGACGATTCTAAGAACACT600               G CCTATTTACAGATCAACAGCCTCAGAGCCGAGGACACGGCTGTGTATTTCTGTACAAGA660              CGGGGTTACGACTGGTACTTCGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA720               TGA 723                                                                       (2) INFORMATION FOR SEQ ID NO:93:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 723 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                      GAGATCCAGTTGGTGCAGTCTGGAGGAGGCCTGGTGAAGCCTGGAG GGTCCGTCAGAATC60               TCCTGCGCAGCTTCTGGGTATACCTTCACAAACTATGGAATGAACTGGGTGCGCCAGGCT120               CCAGGAAAGGGTTTAGAGTGGATGGGCTGGATAAACACCCACACTGGAGAGCCAACATAT180               GCTGATTCTTTCAAGGGACGGTT TACCTTCTCTTTGGACGATTCTAAGAACACTGCCTAT240              TTACAGATCAACAGCCTCAGAGCCGAGGACACGGCTGTGTATTTCTGTACAAGACGGGGT300               TACGACTGGTACTTCGATGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGC360                GGTGGATCTGGTGGAGGTGGGTCCGGAGGTGGAGGATCTGACATCCAGATGACTCAGTCT420              CCATCTTCCCTGTCTGCATCTGTAGGAGACAGAGTCACTATCACTTGCCGGGCGAGTCAG480               GACATTAATAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAAG CTCCTAAGACCCTG540              ATCTATCGTGCAAACAGATTGGAATCTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCT600               GGGACAGATTATACTCTCACCATCAGCAGCCTGCAATATGAAGATTTTGGAATTTATTAT660               TGTCAACAGTATGATGAGTCTCC GTGGACGTTCGGTGGAGGCACCAAGCTTGAGATGAAA720              TGA723                                                                        (2) INFORMATION FOR SEQ ID NO:94:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                      CGGACCCACCTCCACCAGATCCACCGCCACCTTTCATCTCAAGCTTGGTGC51                         (2) INFORMATION FOR SEQ ID NO:95:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                      GACATCCAGATGACTCAGT19                                                         (2) INFORMATION FOR SEQ ID NO:96:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                        (C ) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                      GGTGGAGGTGGGTCCGGAGGTGGAGGATCTGAGATCCAGTTGGTGCAGT49                           (2) INFORMATION FOR SEQ ID NO:97:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 35 base pairs                                                     (B) TYPE: nucleic acid                                                        ( C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                      TGTACTCGAGCCCATCATGAGGAGACGGTGACCGT35                                         (2) INFORMATION FOR SEQ ID NO:98:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                      GGTGGAGGTGGGTCCGGAGGTGGAGGATCT30                                              (2) INFORMATION FOR SEQ ID NO:99:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                      TGTACTCGAGCCCATCATTTCATCTCAAGCTTGGTGC37                                       (2) INFORMATION FOR SEQ ID NO:100:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                                     GAGATCCAGTTGGTGCAGTCTG22                                                      (2) INFORMATION FOR SEQ ID NO:101:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 49 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                     (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                                     CGGACCCACCTCCACCAGATCCACCGCCACCTGAGGAGACGGTGACCGT49                       

We claim:
 1. An isolated polynucleotide encoding an analog of the Type Iribosome inactivating protein, gelonin, wherein a cysteine issubstituted for an amino acid which is not naturally available forintermolecular disulfide bonding in gelonin and said cysteine beingavailable for intermolecular disulfide bonding, wherein said gelonin hasthe amino acid sequence of SEQ ID NO:2 and said cysteine is substitutedfor an amino acid in said gelonin from amino acid 239 to the carboxyterminal amino acid shown in SEQ ID NO: 2, and wherein said analogretains ribosome inactivating activity of said gelonin.
 2. A vectorcomprising the polynucleotide of claim 1 which encodes an analog ofgelonin.
 3. A host cell comprising a vector according to claim
 2. 4. Theisolated polynucleotide recited in claim 1 encoding an analog ofgelonin, wherein said gelonin has the amino acid sequence of SEQ ID NO:2, and wherein said cysteine is substituted for an amino acid at aposition in said amino acid sequence of SEQ ID NO: 2 selected from thegroup consisting of positions 239, 244, 247, and
 248. 5. A vectorcomprising an polynucleotide according to claim
 4. 6. A host cellcomprising the vector according to claim
 5. 7. The host cell recited inclaim 6, wherein said host cell is deposited as ATCC accession No.69009.
 8. The isolated polynucleotide recited in claim 1 encoding ananalog of gelonin, wherein said gelonin has the amino acid sequence ofSEQ ID NO: 2, and wherein additionally the native cysteine residues ofsaid amino acid sequence of SEQ ID NO: 2 at positions 44 and 50 arereplaced with alanine residues.
 9. A polynucleotide encoding an analogof the Type I ribosome inactivating protein, gelonin as shown in SEQ IDNO: 2, wherein a cysteine is substituted in said analog for anotheramino acid at an amino acid position not naturally available forintermolecular disulfide bonding in said gelonin and said cysteine beingavailable for intermolecular disulfide bonding, wherein said cysteine issubstituted at an amino acid position in said gelonin from position 239to the carboxy terminal residue, and wherein said analog retainsribosome-inactivating activity of said gelonin.
 10. A host cellcomprising the vector according to claim
 9. 11. An isolatedpolynucleotide encoding an analog of the Type I ribosome-inactivatingprotein, gelonin, wherein said gelonin has the amino acid sequence ofSEQ ID NO:2 and wherein said analog consists of a cysteine residuesubstituted for a non-cysteine residue at position 60 in the geloninamino acid sequence of SEQ ID NO:
 2. 12. A vector comprising anpolynucleotide according to claim
 11. 13. A host cell comprising thevector according to claim
 12. 14. An isolated polynucleotide encoding ananalog of the Type I ribosome-inactivating protein, gelonin, whereinsaid gelonin has the amino acid sequence of SEQ ID NO:2 and wherein saidanalog consists of a non-cysteine residue substituted for a cysteineresidue at position 50 in the gelonin amino acid sequence of SEQ ID NO:2.
 15. A vector comprising an polynucleotide according to claim
 14. 16.A host cell comprising the vector according to claim
 15. 17. An isolatedpolynucleotide encoding an analog of the Type I ribosome-inactivatingprotein, gelonin, wherein said gelonin has the amino acid sequence ofSEQ ID NO: 2; and wherein a non-cysteine amino acid is substituted atposition 44 in SEQ ID NO:
 2. 18. A vector comprising the polynucleotideaccording to claim
 17. 19. A host cell comprising the vector accordingto claim
 18. 20. An isolated polynucleotide encoding an analog of theType I ribosome-inactivating protein, gelonin, wherein said gelonin hasthe amino acid sequence of SEQ ID NO:2 and wherein said analog consistsof a cysteine residue substituted for a non-cysteine residue at position10 in the gelonin amino acid sequence of SEQ ID NO:
 2. 21. A vectorcomprising the polynucleotide according to claim
 20. 22. A host cellcomprising the vector according to claim
 21. 23. The host cell recitedin claim 22, wherein said host cell is deposited as ATCC Accession No.69008.